Artificial cell comprising mutant estrogen receptor

ABSTRACT

The present invention provides in general an artificial cell, an isolated mutant ERα, an isolated polynucleotide encoding the mutant ERα, a method for quantitatively analyzing an activity for transactivation of a reporter gene by a test ERα, a method for screening a mutant ligand dependent transcriptional factor, a method for evaluating an activity for transactivation of a reporter gene by a test ERα, a method for screening a compound useful for treating a disorder of a mutant ERα, a pharmaceutical agent useful for treating an estrogenic disorder of a mutant ERα, use of the mutant ERα, a method for diagnosing a genotype of a polynucleotide encoding a test ERα, a method for diagnosing a genotype of a polynucleotide encoding a test ERα and a method for diagnosing a phenotype of a test ERα.

This application is the national phase under 35 U.S.C. 371 of PCTInternational Application No. PCT/JP00/08553 which has an Internationalfiling date of Dec. 1, 2000, which designated the United States ofAmerica.

1. TECHNICAL FIELD OF THE INVENTION

The present invention relates to ligand dependent transcriptionalfactors, such as an ERα, and to genes encoding a ligand dependenttranscriptional factor. Further, the present invention relates to cellscontaining a ligand dependent transcriptional factor and a specifiedreporter gene for the ligand dependent transcriptional factor.

2. BACKGROUND OF THE INVENTION

Various cell mechanisms are regulated by ligand dependenttranscriptional factors. The regulation by the ligand dependenttranscriptional factors is usually achieved because the ligand dependenttranscriptional factor has an activity for transactivation of a gene. Ithas been postulated that in transactivation, the ligand dependenttranscription factor and a RNA polymerase II complex interact togetherat a gene to increase the rate of gene expression. The transactivationcan often determine in eukaryotic cells, whether a gene is sufficientlyexpressed to regulate the various cell mechanisms.

Such transactivation by the ligand dependent transcriptional factors canoccur when the ligand dependent tranactivational factor is selectivelybound to its cognate ligand and to its cognate responsive elementsequence. In this regard, the presence of the cognate responsive elementin a gene or the presence of its cognate ligand in the cell candetermine whether the ligand dependent transcriptional factor cantransactivate the gene.

ERα is an example of such ligand dependent transcriptional factors. ERαis naturally found in the target cells of estrogen such as in ovarycells, breast cells, uterus cells, bone cells and the like. Thetransactivation activity of ERα typically occurs when ERα is selectivelybound to an ERE and an estrogen such as E2. It is reported that aberranttransactivation by ERα may contribute to various disorders. Attemptshave been made to use anti-estrogens that are antagonistic to a normalERα. Examples of such anti-estrogens used with such disorders includetamoxifen, raloxifene, 4-hydroxytamoxifen and the like.

3. SUMMARY OF THE INVENTION

The present invention provides in general an artificial cell, anisolated mutant ERα, an isolated polynucleotide encoding the mutant ERα,a method for quantitatively analyzing an activity for transactivation ofa reporter gene by a test ERα, a method for screening a mutant liganddependent transcriptional factor, a method for evaluating an activityfor transactivation of a reporter gene by a test ERα, a method forscreening a compound useful for treating a disorder of a mutant ERα, apharmaceutical agent useful for treating an estrogenic disorder of amutant ERα, use of the mutant ERα, a method for diagnosing a genotype ofa polynucleotide encoding a test ERα, a method for diagnosing a genotypeof a polynucleotide encoding a test ERα and a method for diagnosing aphenotype of a test ERα.

4. DESCRIPTION OF FIGURES

FIGS. 1 to 32 illustrate the luciferase activity provided by a humanmutant ERα or a human normal ERα. The reporter gene was expressed in thechromosomes of the cell. The mutant ERα gene was transiently expressedin the cell. FIGS. 1, 2, 4, 5, 7, 8, 12, 13, 16, 17 and 21 to 26illustrate the luciferase activity in the presence of variousconcentrations of 4-hydroxytamoxifen, raloxifene or ZM189154 as the soleprobable agent of stimulating a human mutant ERα or a human mutant ERα.FIGS. 3, 6, 9 to 11, 14, 15, 18 to 20, 27 to 32 illustrate theluciferase activity in the presence of various concentrations of 100 pMof E2 with various concentrations of 4-hydroxytamoxifen, raloxifene orZM189154. A stable transformed cassette cell was utilized to transientlyexpress the human mutant ERα gene or the human normal ERα gene as wellas to express in a chromosome thereof the reporter gene. FIGS. 1, 2, 4,5, 7, 8, 12, 13, 16, 17 and 21 to 26 zero together the luciferaseactivity by using the luciferase activity provided by the controls, inwhich the human mutant ERα or the human normal ERα was in the presenceof DMSO (containing no 4-hydroxytamoxifen, raloxifene or ZM189154).FIGS. 3, 6, 9-11, 14, 15, 18 to 20, 27 to 32 zero together theluciferase activity by using the luciferase activity provided by thecontrols, in which the human mutant ERα or the human mutant ERα was inthe presence of a DMSO solution containing 100 pM of E2. In zeroing theluciferase activity provided by the human mutant ERα and human normalERα, the luciferase activity by the controls were set together as 100%luciferase activity. The luciferase activity provided by the controls inFIGS. 1, 2, 4, 5, 7, 8, 12, 13, 16, 17 and 21 to 26 is shown as DMSO.The luciferase activity provided by the controls in FIGS. 3, 6, 9-11,14, 15, 18 to 20; 27 to 32 is shown as DMSO+E2.

FIGS. 33 to 48 illustrate the luciferase activity provided by a humanmutant ERα or a human normal ERα. The reporter gene, the human mutantERα gene and the human normal ERα were expressed in the chromosomes ofthe cell. FIGS. 33 to 40 illustrate the luciferase activity in thepresence of various concentrations of 4-hydroxytamoxifen, ZM189154 orraloxifene as the sole probable agent of stimulating a human mutant ERαor a human normal ERα. FIGS. 41 to 48 illustrate the luciferase activityin the presence of 100 pM of E2 with various concentrations of4-hydroxytamoxifen, ZM189154 or raloxifene. A stably transformed binarycell was utilized to express in the chromosomes, the reporter gene withthe human mutant ERα gene or with the human normal ERα gene. FIGS. 33 to40 zero together the luciferase activity provided by the controls, inwhich the human mutant ERα or the human normal ERα was in the presenceof DMSO (containing no 4-hydroxytamoxifen, raloxifene or ZM189154).FIGS. 41 to 48 zero together the luciferase activity provided by thecontrols, in which the human mutant ERα or the human normal ERα was inthe presence of a DMSO solution containing 100 pM of E2. In zeroing theluciferase activity provided by the human mutant ERα and human normalERα, the luciferase activity by the controls were set together as 100%luciferase activity. The luciferase activity provided by the controls inFIGS. 33 to 40 is shown as DMSO. The luciferase activity provided by thecontrols in FIGS. 41 to 48 is shown as DMSO+E2.

FIGS. 49 to 52 illustrate, as a comparative example, the luciferaseactivity provided by a human mutant ERαK531E or a human normal ERα, inwhich the reporter gene was transiently expressed in the cell. FIGS. 49and 50 illustrate the luciferase activity in the presence of variousconcentrations of 4-hydrozytamoxifen as the sole probable agent ofstimulating a human mutant ERα. FIGS. 51 and 52 illustrate theluciferase activity in the presence of 100 pM of E2 with variousconcentrations of 4-hydroxytamoxifen. FIGS. 49 and 50 zero together theluciferase activity of the controls, in which the human normal ERα orthe human mutant ERαK531E was in the presence of DMSO (containing no4-hydrozytamoxifen). FIGS. 51 and 52 zero together the luciferaseactivity of the controls, in which the human normal ERα or the humanmutant ERαK531E was in the presence of a DMSO solution containing 100 pMof E2. In zeroing the luciferase activity provided by the human mutantERα and human normal ERα, the resulting luciferase activity by thecontrols is set as 100% luciferase activity. The luciferase activityprovided by the controls in FIGS. 49 and 50 is shown as DMSO. Theluciferase activity provided by the controls in FIGS. 51 and 52 is shownas DMSO+E2.

5. DETAILED DESCRIPTION OF THE INVENTION 5.1. Definitions

AR: means the androgen receptor protein.

E2: means estradiol.

ERα: means an estrogen receptor α protein. Specified mutants of ERα arereferred to herein by a letter-number-letter combination following thephrase “mutant ERα”, such as by K303R, S309F, G390D, M396V, G415V,G494V, K531E and S578P. In the letter-number-letter combination, thenumber indicates the relative position of a substituted amino acid inthe mutant ERα, the letter preceding the number indicates the amino acidin a normal ERα at the indicated relative position and the letterfollowing the number indicates the substituted amino acid in theprovided mutant ERα at the indicated relative position. When there aretwo substituted amino acids in the mutant ERα, the phrase “mutant ERα”is followed by two letter-number-letter combinations, such as byG390D/S578P.

ERβ: means the estrogen receptor β protein.

GR: means the glucocorticoid receptor protein.

MR: means the mineralocorticoid receptor protein.

PPAR: means the peroxisome proliferator-activated receptor protein.

PR: means the progesterone receptor protein.

PXR: means the pregnane X receptor protein.

TR: means the thyroid hormone receptor protein.

VDR: means the vitamin D receptor protein.

DR1: means the receptor responsive sequence having the followingnucleotide sequence:

-   -   5′-AGGTCAnAGGTCA-3′        wherein n represents an A, C, T or G.

DR3: means the receptor responsive sequence having the followingnucleotide sequence:

-   -   5′-AGGTCAnnnAGGTCA-3′        wherein n represents an A, C, T or G.

DR4: means the receptor responsive sequence having the followingnucleotide sequence:

-   -   5′-AGGTCAnnnnAGGTCA-3′        wherein n represents an A, C, T or G.

ERE: means the estrogen responsive element nucleotide sequence.

MMTV: means the mouse mammary tumor virus

5.2. The Cell

The cell of the present invention comprises a chromosome which comprisesa reporter gene. The reporter gene in the chromosome comprises an ERE, aTATA sequence and a reporter sequence. In addition, the cell comprises amutant ERα or a gene encoding the mutant ERα. In this regard, the cellprovides a biological system in which the mutant ERα can have anactivity for transactivation of the reporter gene. The activity fortransactivation of the reporter gene by the mutant ERα in the presenceof E2 and a partial anti-estrogen is typically higher than that by anormal ERα in the presence of E2 and the partial anti-estrogen.Alternatively, the activity for transactivation of the reporter gene bythe mutant ERα in the presence of the partial anti-estrogen as the soleprobable agent of stimulating the mutant ERα is typically higher thanthat by the normal ERα in the presence of the partial anti-estrogen asthe sole probable agent of stimulating the normal ERα.

Typically, the ERE, the TATA sequence and the reporter sequence areorganized in the reporter gene to allow the transactivation of thereporter gene. For example, the reporter gene can have the ERE operablyupstream from the TATA sequence and the reporter sequence operablydownstream from the TATA sequence. If so desired, the reporter gene mayadditionally contain conventional nucleotide sequences advantageous forthe expression of the reporter gene.

The TATA sequence may have the following nucleotide sequence:

-   -   5′-TATAA-3′

In a natural cell, the ERE is a receptor responsive sequence that iscognate with a normal ERα. When normal ERα binds to E2 and the normalERα-E2 complex binds to the ERE, the normal ERα has an activity fortransactivation. In the cell, it is a function of the ERE to bind to themutant ERα and allow the mutant ERα to have an activity fortransactivation of the reporter gene. Typically, such an ERE isencompassed by the following nucleotide sequence:

-   -   5′-AGGTCAnnnTGACCTT-3′        wherein n represents an A, G, C or T. Further, a tandem repeat        of the ERE in the reporter gene can provide a more efficient        activity for transactivation of the reporter gene. A 2 to 5        tandem repeat of the ERE may be used in the reporter gene. As an        example of an ERE which can be utilized in the reporter gene,        there is mentioned an ERE derived from Xenopus vitellogenin gene        (Cell, 57, 1139-1146). The ERE can be prepared for the reporter        gene by being chemically synthesized or by being cloned with        polymerase chain reaction (PCR) amplification methods.

The reporter sequence in the reporter gene is a reporter sequencenaturally foreign to the ERE. As such, the reporter sequence and the EREare not found together in a natural gene. Further, when such a reportersequence encodes a reporter protein, the reporter sequence typicallyencodes a reporter protein that is more or less active in the cell. Asexamples of the reporter protein, there is mentioned a luciferase, asecretory alkaline phosphatase, a β-galactosidase, a chloramphenicolacetyl transferase, a growth hormone and the like.

Conventional methods may be used to ligate the ERE, the TATA sequenceand the reporter sequence. After producing the reporter gene, thereporter gene may be inserted into a chromosome. The reporter gene maybe inserted into a chromosome when the reporter gene is introduced intoa host cell. Such methods of introducing the reporter gene into a hostcell are described below.

The mutant ERα in the cell typically has a particular activity fortransactivation of the reporter gene when in the presence of E2 and apartial anti-estrogen or when in the presence of the partialanti-estrogen as the sole probable agent of stimulating the mutant ERα.The activity for transactivation provided by the mutant ERα in thepresence of E2 and the partial anti-estrogen is typically higher thanthat by a normal ERα in the presence of E2 and the partialanti-estrogen. The activity for transactivation of the reporter gene bythe mutant ERα in the presence of the partial anti-estrogen as the soleprobable agent of stimulating the mutant ERα is higher than that by thenormal ERα in the presence of the partial anti-estrogen as the soleprobable agent of stimulating the normal ERα. Since transactivationinvolves the increase of rate of transcription, such a transactivationby the normal ERα and mutant ERα can be observed by measuring theexpression level of the reporter gene. When the expression levels of thereporter gene provided by the mutant ERα and normal ERα are adjusted tobe zeroed at identical points, the mutant ERα would provide a higherexpression level than that provided by the normal ERα.

Further, it should be noted that the mutant ERα may have the activityfor transactivation of the reporter gene inhibited in the presence ofthe pure anti-estrogen. Such a activity for transactivation for thereporter gene provided by the mutant ERα is similar to the inhibition ofthe activity for transactivation of the reporter gene provided by thenormal ERα in the presence of the pure anti-estrogen.

A normal ERα encompasses the ERα which is reported as most commonlycarried in a species, such as human, monkey, mouse, rabbit, rat and thelike. For example, a human normal ERα has the amino acid sequence shownin SEQ ID:1. Such a human normal ERα is described in Tora L. et al.,EMBO, vol 8 no 7: 1981-1986 (1989).

The partial anti-estrogens typically are not antagonistic to an AF1region of the normal ERα and are antagonistic to an AF2 region of anormal ERα. The AF2 region of a normal ERα and the AF1 region of anormal ERα are each regions in the normal ERα that are involved intransactivation by the normal ERα (Metzger D. et al., J. Biol. Chem.,270:9535-9542 (1995)).

Such properties of the partial anti-estrogens may be observed, forexample, by carrying out the reporter assay described in Berry M. etal., EMBO J., 9:2811-2818 (1990). In such a reporter assay, there isutilized cells in which the AF1 region of an endogenous normal ERα has astrong activity for transactivation, such as chicken embryo fibroblastcells in primary culture (that may be prepared according to thedescription, for example, in Solomon, J. J., Tissue Cult. Assoc.Manual., 1:7-11 (1975)). When utilized, the chicken embryo fibroblastare modified so that the modified fibroblasts express therein a geneencoding the normal ERα and so that the modified fibroblasts have thereporter gene (hereinafter referred to as AF1 evaluation fibroblasts).When the AF1 evaluation fibroblasts are exposed with a sufficient amountof a partial anti-estrogen, it can be determined whether the partialanti-estrogen fails to be antagonistic to an AF1 region of a normal ERα.The partial anti-estrogen in such cases increase the expression level ofthe reporter gene in the AF1 evaluation fibroblasts. Further, thechicken embryo fibroblast cells in primary culture are then modified fora second round so that the second modified fibroblasts express a geneencoding a truncated normal ERα which has the AF1 region deleted and sothat the second modified fibroblasts have the reporter gene (hereinafterreferred to as AF2 evaluation fibroblasts). When the AF2 evaluationfibroblasts are exposed with a sufficient amount the partialanti-estrogen, it can be determined whether the partial anti-estrogen isantagonistic to an AF2 region of a normal ERα. The partial anti-estrogenin such cases fails to increase the expression level of the reportergene in the AF2 evaluation fibroblasts.

Examples of such parital anti-estrogens include tamoxifen,4-hydroxytamoxifen, raloxifene and the like.

The pure anti-estrogen is typically an anti-estrogen which is fullyantagonistic to a normal ERα. In this regard, the pure anti-estrogenfails to be partially agonistic to the ERα. In a reporter assay witheither the AF1 evaluation fibroblasts or the AF2 evaluation fibroblasts,the pure anti-estrogen provides substantially no activity fortransactivation of the reporter gene by the normal ERα or truncatednormal ERα therein. As such, the expression level of the reporter genein such reporter assays with the pure-anti-estrogen and either of theAF1 evaluation fibroblasts or AF2 evaluation fibroblasts does notsubstantially increase.

Examples of such pure anti-estrogen include ICI 182780 (Wakeling A E etal., Cancer Res., 512:3867-3873 (1991)), ZM 189154 (Dukes M et al., J.Endocrinol., 141:335-341 (1994)) and the like.

The mutant ERα comprises one or more substituted amino acids whichconfers such an activity for transactivation of the reporter gene in thepresence of E2 and the partial anti-estrogen or in the presence of thepartial anti-estrogen as the sole probable agent of stimulating themutant ERα. Typically, the one or more substituted amino acids arepresent in the mutant ERα at one or more relative positions of from 303to 578. For example, the mutant ERα may comprise one or more substitutedamino acids at one or more relative positions selected from 303, 309,390, 396, 415, 494, 531, 578 and the like. Typically, such relativepositions in the mutant ERα are based on a homology alignment to theamino acid sequence shown in SEQ ID:1.

In general, a homology alignment encompasses an alignment of amino acidsequences based on the homology of the provided amino acid sequences.For example, Table 1 below randomly sets forth a homology alignment withthe amino acid sequence shown in SEQ ID:1 (a human normal ERα), a mouseERα (Genbank Accession No. M38651), a rat ERα(X6) (Genbank Accession No.X61098) and a rat ERα(Y0) (Genbank Accession No. Y00102).

TABLE 1

In Table 1, “hERa.TXT” sets forth the amino acid sequence shown in SEQID:1. “mER.TXT” sets forth the amino acid sequence of the mouse ERα,“ratER(X6).TXT” sets forth the amino acid sequence of the rat ERα(X6)and “ratER(Y0)” sets forth the amino acid sequence of rat ERα(Y0),wherein amino acids sequences thereof are set forth using one letterabbreviations of the amino acids. This alignment was prepared using acommercially available software GENETYX-WIN SV/R ver. 4.0 (SoftwareDevelopment Co.). The symbol “*” indicates the amino acids located atrelative positions 303 and 578.

The relative positions under the homology alignment correspond to theabsolute positions of the amino acid sequence shown in SEQ ID:1. Forexample, relative position 303 encompasses under the homology alignment,the amino acid in the mutant ERα aligned with the 303rd amino acid fromthe N-terminus in the amino acid sequence shown in SEQ ID:1. Further, arelative position 578 encompasses under the homology alignment, theamino acid in the mutant ERα aligned with the 578th amino acid from theN-terminus in the amino acid sequence shown in SEQ ID:1. In reference toTable 1, examples of relative position 303 include the lysine that isthe 303rd amino acid from the amino terminus in the amino acid sequenceshown in SEQ ID:1, the lysine that is the 307th amino acid from theamino terminus in the amino acid sequence of the mouse ERα, the lysinethat is the 308th amino acid from the amino terminus in the amino acidsequence of rat ERα(X6) and the lysine that is the 308th amino acid fromthe amino terminus in the amino acid sequence of the rat ERα(Y0).Further, examples of the relative position 578 in reference to Table 1include the serine that is the 578th amino acid from the amino terminusin the amino acid sequence shown in SEQ ID NO: 1, the serine that is the582th amino acid from the amino terminus in the amino acid sequence ofthe mouse ERα, the serine that is the 583th amino acid from the aminoterminus in the amino acid sequence of the rat ERα(X6) and the serinethat is the 583th amino acid from the amino terminus in the amino acidsequence of rat ERα(Y0).

In this regard, the homology alignment in connection with the presentinvention aligns the amino acid sequence shown in SEQ ID:1 with an aminoacid sequence encoding mutant ERα, based on the homology of the mutantERα and the amino acid sequence shown SEQ ID:1. When aligning the aminoacid sequence of a mutant ERα in the homology alignment to amino acidsequence SEQ ID:1, such a mutant ERα typically has at least an 80%homology with the amino acid sequence shown in SEQ ID:1.

The mutant ERα can be derived from an animal such as a manual. Examplesof such mammals include human, monkey, rabbit, rat, mouse and the like.For the human mutant ERα, the mutant ERα generally has an amino acidlength of 595 amino acids.

In having the substituted amino acid at relative position 303, themutant ERα may be derived from changing the lysine present at relativeposition 303 in a normal ERα into a substituted amino acid. In suchcases, the mutant ERα may have the substituted amino acid at relativeposition 303 be arginine, such as a mutant ERα K303R. The human mutantERα K303R has the amino acid sequence shown in SEQ ID:2.

In having the substituted amino acid at relative position 309, themutant ERα may be derived from changing the serine present at relativeposition 309 in a normal ERα into a substituted amino acid. In suchcases, the mutant ERα may have the substituted amino acid at relativeposition 309 be phenylalanine, such as a mutant ERα S309F. The humanmutant ERα S309F has the amino acid sequence shown in SEQ ID:3.

In having the substituted amino acid at relative position 390, themutant ERα may be derived from changing the glycine present at relativeposition 390 in a normal ERα into a substituted amino acid. In suchcases, the mutant ERα may have the substituted amino acid at relativeposition 390 be aspartic acid, such as a mutant ERα G390D. The humanmutant ERα G390D has the amino acid sequence shown in SEQ ID:4.

In having the substituted amino acid at relative position 396, themutant ERα may be derived from changing the methionine present atrelative position 396 in a normal ERα into a substituted amino acid. Insuch cases, the mutant ERα may have the substituted amino acid atrelative position 396 be valine, such as a mutant ERα M396V. The humanmutant ERα M396V has the amino acid sequence shown in SEQ ID:5.

In having the substituted amino acid at relative position 415, themutant ERα may be derived from changing the glycine present at relativeposition 415 in a normal ERα into a substituted amino acid. In suchcases, the mutant ERα may have the substituted amino acid at relativeposition 415 be valine, such as a mutant ERα G415V. The human mutant ERαG415V has the amino acid sequence shown in SEQ ID:6.

In having the substituted amino acid at relative position 494, themutant ERα may be derived from changing the glycine present at relativeposition 494 in a normal ERα into a substituted amino acid. In suchcases, the mutant ERα may have the substituted amino acid at relativeposition 494 be valine, such as a mutant ERα G494V. The human mutant ERαG494V has the amino acid sequence shown in SEQ ID:7.

In having the substituted amino acid at relative position 531, themutant ERα may be derived from changing the lysine present at relativeposition 531 in a normal ERα into a substituted amino acid. In suchcases, the mutant ERα may have the substituted amino acid at relativeposition 531 be glutamic acid, such as a mutant ERα K531E. The humanmutant ERα K531E has the amino acid sequence shown in SEQ ID:8.

In having the substituted amino acid at relative position 578, themutant ERα may be derived from changing the serine present at relativeposition 578 in a normal ERα into a substituted amino acid. In suchcases, the mutant ERα may have the substituted amino acid at relativeposition 578 be proline, such as mutant ERα S578P. The human mutant ERαS578P has the amino acid sequence shown in SEQ ID:9.

In having the substituted amino acid at relative position 390 and 578,the mutant ERα may be derived from changing the glycine present atrelative position 390 in a normal ERα into a substituted amino acid aswell as changing the serine present at relative position 578 in thenormal ERα into another substituted amino acid. In such cases, themutant ERα may have the substituted amino acid at relative position 390be aspartic acid and the substituted amino acid at relative position 578be proline, such as mutant ERα G390D/S578P. The human mutant ERαG390D/S578P has the amino acid sequence shown in SEQ ID:10.

To provide the mutant ERα, the cell may express a gene encoding themutant ERα, according to the standard genetic code which is well known.Such a mutant ERα gene typically comprises a polynucleotide whichencodes the mutant ERα and a promoter. The mutant ERα gene can beisolated from tissue sample. Further, the mutant ERα gene may beproduced by using mutagenesis techniques to mutagenize a polynucleotideencoding a normal ERα to encode the mutant ERα and by operably linking apromoter upstream from the resulting polynucleotide encoding the mutantERα. The mutagenesis techniques, such as site-directed mutagenesis, maybe utilized to introduce the one or more mutations into the normal ERαpolynucleotide and provide a mutant ERα polynucleotide. The human normalERα polynucleotide having the nucleotide sequence described in Tora L.et al. EMBO J., vol 8 no 7:1981-1986 (1989) is utilized in the case ofmutagenizing the human normal ERα polynucleotide.

The promoter in the mutant ERα gene initiates transcription so that themutant ERα can be expressed to provide the mutant ERα in the cell. Inthis regard, a promoter capable of functioning in the cell is usuallyoperably linked upstream to a polynucleotide encoding the mutant ERα.For instance, where the cell is derived from an animal host cell orfission yeast host cell, examples of the promoter may include Roussarcoma virus (RSV) promoter, cytomegalovirus (CMV) promoter, early andlate promoters of simian virus (SV40), MMTV promoter and the like. Wherethe cells are derived from budding yeast host cell, examples of thepromoter may include ADH1 promoter and the like.

In using the mutagenesis techniques, a polynucleotide encoding normalERα can be isolated and then the isolated normal ERα polynucleotide canbe mutagenized by using oligonucleotides. The resulting mutant ERαpolynucleotide can then be utilized to produce the mutant ERα gene.

Oligonucleotides are designed and synthesized to specifically amplify acDNA encoding a normal ERα from a cDNA library or the cDNAs of ananimal. Such oligonucleotides can be designed, based on a well knownnucleotide sequence encoding the normal ERα, such as the normal ERαnucleotide sequences found in documents, such as Tora L. et al. EMBO J.,vol 8 no 7:1981-1986 (1989), or in databases such as in Genbank. As suchnormal ERα nucleotide sequences, there can be utilized a normal ERαnucleotide sequence derived from human, monkey, rabbit, rat, mouse orthe like. The designed oligonucleotides can then be synthesized with aDNA synthesizer (Model 394, Applied Biosystems). A polymerase chainreaction (PCR) amplification may then be utilized to isolate the normalERα polynucleotide from the cDNA library or cDNAs. For human normal ERαgene, the oligonucleotides depicted in SEQ ID:11 and SEQ ID:12 may beutilized to PCR amplify the human normal ERα polynucleotide having thenucleotide sequence described in Tora L. et al. EMBO J., vol 8 no7:1981-1986 (1989).

The cDNAs can be derived from animal tissue (such as human, monkey,rabbit, rat, or mouse) according to genetic engineering techniquesdescribed in J. Sambrook, E. F. Frisch, T. Maniatis, “Molecular Cloning,2nd edition”, Cold Spring Harbor Laboratory, 1989. In such techniques,the RNAs in an animal tissue, such as liver or uterus, are collectivelyextracted therefrom and the RNAs are collectively reverse transcribedinto the cDNAs of the animal. For example, the animal tissue is firsthomogenized in a buffer containing a protein denaturing agent such asguanidine hydrochloride or guanidine thiocyanate. Reagents such as amixture containing phenol and chloroform (hereinafter referred to asphenol-chloroform) are further added to denature proteins resulting fromhomogenizing the animal tissue. After removing the denatured proteins bycentrifugation, the RNAs are collectively extracted from the recoveredsupernatant fraction. The RNAs can be collectively extracted by methodssuch as the guanidine hydrochloride/phenol method, SDS-phenol method,the guanidine thiocyanate/CsCl method and the like. ISOGEN (Nippon Gene)is an example of a commercially available kit which is based on suchmethods of collectively extracting the RNAs. After collectivelyextracting the RNAs, oligo-dT primers are allowed to anneal to the polyA sequence in the RNAs to collectively reverse transcribe the RNAs as atemplate. A reverse transcriptase can be utilized to collectivelyreverse transcribe the RNAs into single-stranded cDNAs. The cDNAs can besynthesized from the single-strand cDNAs by using E. coli DNA polymeraseI with the above single-stranded cDNAs. In using E. coli DNA polymeraseI, E. coli RNase H is also used to produce primers, which allow E. coliDNA polymerase I to operate more efficiently. The cDNAs can be purifiedby using conventional purifying procedures, for example, byphenol-chloroform extraction and ethanol precipitation. Examples ofcommercially available kits based on such methods include cDNA SynthesisSystem Plus (Amersham Pharmacia Biotech), TimeSaver cDNA Synthesis kit(Amerham Pharmacia Biotech) and the like.

The normal ERα polynucleotide is then isolated from the cDNAs. Isolationprocedures which may be utilized to isolate the normal ERαpolynucleotide may include using PCR amplification. The PCRamplification typically amplifies the normal ERα polynucleotide from thecDNAs. The PCR mixture in the PCR amplification may contain a sufficientamount of the cDNAs, a sufficient amount of the forward and reverseoligonucleotides, a heat tolerant DNA polymerase (such as LT-Taqpolymerase (Takara Shuzo)), dNTPs (dATP, dTTP, dGTP, dCTP) and a PCRamplification buffer. In a PCR mixture amplifying a human normal ERαpolynucleotide, there may be utilized 10 ng of the cDNAs and 10 pmol ofthe each of the forward and reverse oligonucleotides (SEQ ID:11 and SEQID:12). The PCR mixture in the PCR amplification then undergoes anincubation cycle for annealing, elongation and denaturing. For example,the PCR amplification may have repeated 35 times with a thermal cyclersuch as PCR System 9700 (Applied Biosystems), an incubation cycleentailing an incubation at 95° C. for 1 minute and then an incubation at68° C. for 3 minutes. After the PCR amplification with the cDNAs, thewhole amount of resulting PCR mixture is subjected to low melting pointagarose gel electrophoresis (Agarose L, Nippon Gene). After confirmingthe presence of a band therein comprising the normal ERα polynucleotide,the normal ERα polynucleotide is recovered from the low melting pointagarose gel.

As the cDNA libraries, there can be utilized a commercially availablecDNA library derived from an animal, such as QUICKClone cDNAs(manufactured by Clontech). The cDNA library may then be isolated asdescribed above.

The nucleotide sequence of the recovered normal ERα polynucleotide canbe confirmed by preparing a sample of the normal ERα polynucleotide fordirect sequencing. Also, DNA fluorescence sequencing techniques may beutilized to sequence the normal ERα polynucleotide. In this regard, toprepare the sample of the normal ERα polynucleotide, there can beutilized commercially available reagents for fluorescence sequencingsuch as Dye Terminator Sequencing kit FS (Applied Biosystems). Thefluorescence sequencing of the normal ERα polynucleotide may beconducted with an autosequencer such as ABI autosequencer (Model 377,Applied Biosystems). Further, the normal ERα polynucleotide may bemanually sequenced (Biotechniques, 7, 494(1989)).

For convenience, the isolated normal ERα polynucleotide can be insertedinto a vector capable of replicating in a host such as E. coli. Forexample, about 1 μg of isolated normal ERα polynucleotide may have theends thereof blunted by a treatment with DNA blunting kit (TakaraShuzo), when the provided isolated normal ERα polynucleotide has unevenends. A T4 polynucleotide kinase may then be used to phosphorylate theends of the blunt-ended normal ERα polynucleotide. After phenoltreatment, the normal ERα polynucleotide is purified by ethanolprecipitation and may be inserted into a vector capable of replicationin E. coli. The E. coli vector comprising the normal ERα polynucleotidemay be cloned into E. coli host cells.

The E. coli vector comprising the normal ERα polynucleotide may then beisolated from the cloned E. coli cells. The isolated E. coli vectorcomprising the normal ERα polynucleotide is then used as a template tomutagenize, i.e., introduce nucleotide substitutions, into the normalERα polynucleotide, such that the resulting mutant ERα polynucleotidecontains a variant codon encoding the substituted amino acid at thedesired relative position.

The desired nucleotide substitutions may be introduced into the normalERα polynucleotide according to the site-directed mutagenesis methodsdescribed in J. Sambrook, E. F., Frisch, T. Maniatis, “Molecular Cloning2nd edition”, Cold Spring Harbor Laboratory, 1989, or the site directedmutagenesis methods described in McClary J A et al., Biotechniques1989(3): 282-289. For example, the desired nucleotide substitutions maybe introduced into the normal ERα polynucleotide by using a commerciallyavailable kit, such as QuickChange Site-Directed Mutagenesis kitmanufactured by Stratagene. Typically, such site-directed mutagenesismethods utilize oligonucleotides which introduce the desired nucleotidesubstitutions therein. In relation to the QuickChange Site-DirectedMutagenesis kit, the following describes in more detail thesite-directed mutagenesis methods utilized with the normal ERαpolynucleotide.

The QuickChange Site-Directed Mutagenesis kit utilizes twooligonucleotides to achieve the desired nucleotide substitution into thenormal ERα polynucleotide. As such a combination of the twooligonucleotides, there may be utilized for human normal ERαpolynucleotide, the combination of oligonucleotides selected from thecombination including the oligonucleotide depicted in SEQ ID:13 with theoligonucleotide depicted in SEQ ID:14, the combination including theoligonucleotide depicted in SEQ ID:15 with the oligonucleotide depictedin SEQ ID:16, the combination including the oligonucleotide depicted inSEQ ID:17 with the oligonucleotide depicted in SEQ ID:18, thecombination including the oligonucleotide depicted in SEQ ID:19 with theoligonucleotide depicted in SEQ ID:20, the combination including theoligonucleotide depicted in SEQ ID:21 with the oligonucleotide depictedin SEQ ID:22, the combination including the oligonucleotide depicted inSEQ ID:23 with the oligonucleotide depicted in SEQ ID:24, thecombination including the oligonucleotide depicted in SEQ ID:25 with theoligonucleotide depicted in SEQ ID:26 or the combination including theoligonucleotide depicted in SEQ ID:27 with the oligonucleotide depictedin SEQ ID:28. Table 2 below shows the relative position of the aminoacid encoded at the locus of the nucleotide substitution and theresulting variant codons from utilizing such combinations of theoligonucleotides.

TABLE 2 SEQ ID of oligo- relative nucleotide sequence in variant codonin nucleotides position encoding normal ERα encoding mutant ERα 13 & 14303 AAG (lysine) AGG (arginine) 15 & 16 309 TCC (serine) TTC(phenylalanine) 17 & 18 390 GGT (glycine) GAT (aspartic acid) 19 & 20396 ATG (methionine) GTG (valine) 21 & 22 415 GGA (glycine) GTA (valine)23 & 24 494 GGC (glycine) GTC (valine) 25 & 26 531 AAG (lysine) GAG(glutamic acid) 27 & 28 578 TCC (serine) CCC (proline)The achieved mutant ERα polynucleotide can be sequenced to confirm thatthe desired nucleotide substitution has been introduced into the normalERα polynucleotide.

To produce the cell, the mutant ERα gene and the reporter gene areusually introduced into a host cell. The reporter gene is introducedinto the host cell so that the reporter gene is inserted into achromosome of the host cell. The mutant ERα gene is introduced into thehost cell for transient expression or is inserted into a chromosome ofthe host cell. When inserting the mutant ERα gene into a chromosome ofthe host cell, the mutant ERα gene and reporter gene may be introducedinto one chromosome or the mutant ERα gene may be inserted intochromosome other than the chromosome utilized for the reporter gene.

The host cell typically fails have an expressed normal or mutant ERα.Examples of the host cells may include budding yeast cells such asCG1945 (Clontech), animal cells such as HeLa cells, CV-1 cells, Hepa1cells, NIH3T3 cells, HepG2 cells, COS1 cells, BF-2 cells, CHH-1 cellsand insect cells and the like.

The mutant ERα gene and the reporter gene may be inserted into vectors,so that the mutant ERα gene and the reporter gene can be introduced intothe host cell. Such vectors typically have a replication origin so thatthe vector can be replicated in the cell. If so desired, the vector mayalso have a selective marker gene.

Where the budding yeast cell is used as a host cell, examples of thevector may include plasmid pGBT9, pGAD424, pACT2 (Clontech) and thelike. Where mammalian cells are used as host cells, examples of thevector may include plasmids such as pRc/RSV, pRc/CMV (Invitrogen),vectors containing an autonomous replication origin derived from virusessuch as bovine papilloma virus plasmid pBPV (Amersham PharmaciaBiotech), EB virus plasmid pCEP4 (Invitrogen) and the like.

When producing a vector encoding the mutant ERα (hereinafter referred toas the mutant ERα vector), it is preferable for the vector toadditionally contain the promoter so that the mutant ERα polynucleotidecan be inserted into the vector to produce together the mutant ERα genewith the mutant ERα vector. Likewise, when producing a vector encodingthe reporter gene (hereinafter referred to as the reporter vector), itis preferable for the vector to contain a TATA sequence or an ERE sothat the reporter gene can be produced together with the reportervector.

When producing the mutant ERα vector together with the mutant ERα genefor an animal host cell, pRc/RSV or pRc/CMV can be utilized. Theplasmids pRc/RSV and pRc/CMV contain a promoter which can function inthe cell, when derived from an animal host cell, and a cloning citeoperably downstream from the promoter. In this regard, the mutant ERαvector can be produced together with the mutant ERα gene by insertingthe mutant ERα polynucleotide into pRc/RSV or pRc/CMV at the cloningsite. Since pRc/RSV and pRc/CMV also contain an autonomous replicationorigin of SV40 (ori), pRc/RSV and pRc/CMV may be used to introduce themutant ERα gene into the animal host cells transformed with ori(−) SV40genome, if so desired. As such animal host cells transformed with ori(−)SV40 genome, there is mentioned COS cells. When introduced into suchanimal host cells transformed with ori(−) SV40 genome, the mutant ERαvector produced from pRc/RSV or pRc/CMV can increase to a fairly largecopy number therein such that the mutant ERα gene can be expressed in alarge amount.

When introducing the mutant ERα vector into a budding yeast host cell,it is preferable to utilize pACT2 to produce the mutant ERα vector.Since pACT2 carries an ADH1 promoter, the mutant ERα gene can beproduced together with the mutant ERα vector by inserting the mutant ERαpolynucleotide downstream of the ADH1 promoter. In such cases, a themutant ERα vector can express the mutant ERα gene in a large amount.

Conventional techniques can be used for introducing the mutant ERα gene,according to the type of host cell. For example, the calcium phosphatemethod, DEAE-dextran method, electroporation, lipofection or the likemay be use where mammalian or insect cells are used as host cells. Whereyeast cells are used as host cells, there may be used a lithium methodsuch as a method using the Yeast transformation kit (Clontech) or thelike.

Furthermore, where the mutant ERα gene is introduced into the host cellas viral DNA, the mutant ERα gene may be introduced into host cells notonly by the techniques as described above, but also by infecting thehost cells with recombinant virions containing the vital forms of thereporter gene and the mutant ERα gene. For example, viruses such asvaccinia virus may utilized for animal host cells and where insectanimal cells are used as host cells, there may be utilized insectviruses such as baculovirus.

When the mutant ERα vector or the reporter vector comprises theselective marker gene, as described above, the selective marker gene maybe employed to clone the cell of the present invention. In such cases,the selective marker gene can be utilized to confer a drug resistance toa selective drug exhibiting lethal activity on the cell. The cell inthis regard may then be cloned by culturing the cell in a mediumsupplemented with said selective drug. Exemplary combinations of theselective marker gene and selective drug include a combination ofneomycin resistance-conferring selective marker gene and neomycin, acombination of hygromycin resistance-conferring selective marker geneand hygromycin, a combination of blasticidin S resistance-conferringselective marker gene and blasticidin S and the like. In a case whereinthe selection marker gene encodes a nutrient which complements theauxotrophic properties of the cell, the cell may be cultured using aminimal medium that substantially contains none of the nutrient.Furthermore, an assay measuring an estrogen binding activity may alsoused to clone the cell.

In introducing the reporter gene into the host cell, the reporter geneis usually introduced in a linearized form. The linearized reporter genemay allow the reporter gene to be inserted into the chromosome of thehost cell. When utilizing the reporter vector, the reporter vector canbe linearized by a restriction digestion. The lipofection method may beutilized to introduce the linearized reporter gene into the host cell.

Further, it should be noted that the reporter gene may be introducedinto the host cell, before introducing the mutant ERα gene to provide astably transformed cassette cell. The stably transformed cassette cellstably comprises the reporter gene in a chromosome thereof such that thereporter gene can be genetically handed down to progeny generations. Toproduce the stably transformed cassette cell, the reporter gene may beintroduced into the chromosome of a host cell and the host cell may becultured for several weeks. After culturing for several weeks, thestably transformed cassette cell can be cloned by employing theselective marker gene, when utilized. For example, the transformed hostcells may be continuously cultured for several weeks in a mediumsupplemented with the selective drug to clone the stable transformedcassette cell. The mutant ERα gene may then be introduced into thestably transformed cassette cell to produce the cell.

Furthermore, the mutant ERα gene may also be introduced into the hostcell with the reporter gene so that the host cell is stably transformedwith the reporter gene and the mutant ERα gene.

The cell can be utilized to screen for a compound useful for treating adisorder of the mutant ERα. Such a disorder of a mutant ERα may be adisorder which involves an aberrant transactivation by the mutant ERα,such as breast cancer. To screen such a compound, the cell is exposedwith an efficient amount of a test compound suspected of beingantagonistic or agonistic to the mutant ERα and the transactivationlevel of the reporter gene is measured.

The cell is typically exposed with a sufficient amount of the testcompound for one to several days. The cell can be exposed with the testcompound under agonistic conditions or antagonistic conditions directedto the mutant ERα. The agonistic conditions typically have the assaycell exposed to the test compound as the sole agent probable ofstimulating the mutant ERα. The antagonistic conditions typically havethe assay cell exposed to the test compound and E2.

After exposure, the transactivation level of the reporter gene ismeasured by measuring the expression level of the reporter gene. In suchcases, the reporter protein or the reporter RNA (encoded by the reportersequence) is stored in the cell or is secreted from the cell so that theexpression level can be measured therewith. The expression level of thereporter gene can be measured by a Northern blot analysis, by a Westernblot analysis or by measuring the activity level of the reporterprotein. The activity level of the reporter protein typically indicatesthe level at which the reporter gene is expressed.

For example, when the reporter gene encodes luciferase as the reporterprotein, the expression level of the reporter gene can be measured bythe luminescence provided by reacting luciferin and luciferase. In suchcases, a crude cell extract is produced from the cells and luciferin isadded to the crude cell extract. The luciferin may be allowed to reactwith the luciferase in the cell extract at room temperature. Theluminescence from adding luciferin is usually measured as an indicatorof the expression level or the reporter gene, since the crude cellextract produces a luminescence at a strength proportional to the levelof luciferase expressed in the cell and present in the crude cellextract. A luminometer may be utilized to measure the luminescence inthe resulting crude cell extract.

The measured transactivation level can then be compared with a controlto evaluate the agonistic or antagonistic effect of the test compound.Such a control in screening the test compound can be the expectedtransactivation level of the reporter gene when the cell is not exposedto the test compound. When the transactivation level of the reportergene by the mutant ERα is higher than the control under the agonisticconditions, the test compound is evaluated as an agonist directed to themutant ERα.

Alternatively, when the cell is exposed to E2 and the test compoundunder the antagonistic conditions, the test compound can be evaluated asan antagonist directed to the mutant ERα. In such cases, the control canbe the expected transactivation level of the reporter gene by the mutantERα in the presence of an equivalent amount of E2. When thetransactivation level of the reporter gene by the mutant ERα is lowerthan the control, the test compound is evaluated as being antagonisticto the mutant ERα.

Such a test compound agonistic or antagonistic to the mutant ERα canthen be selected as a compound useful for treating a disorder of themutant ERα. In such cases, the test compound which provides antransactivation level of the reporter gene which is significantly higherthan the control is usually selected when the cell is exposed under theagonistic conditions. The test compound which provides antransactivation level of the reporter gene which is significantly lowerthan the control is usually selected when the cell is exposed under theantagonistic conditions.

Furthermore, compounds for treating disorders of normal ligand dependenttranscription factors can be screened. In such cases, a gene encodingthe normal ligand dependent transcription factor, instead of the mutantERα gene, is introduced into the host cell. Examples of such normalligand dependent transcription factors include a normal ERβ (GenbankAccession No. AB006590), a normal AR (Genbank Accession No. M23263), anormal GR (Genbank Accession No. M10901), a normal TRα (M24748), anormal PR (Genbank Accession No. 15716), a normal PXR (Genbank AccessionNo. AF061056), a normal lipophilic vitamin receptor such as a normal VDR(Genbank Accession No. J03258), a normal RAR (Genbank Accession No.06538), a normal MR (Genbank Accession No. M16801), a normal PPAR γ(Genbank Accession No. U79012) and the like. The reporter gene in suchcases comprises an appropriate receptor responsive sequence cognate withthe normal ligand dependent transcriptional factor, instead of the ERE.

5.3. The Diagnosis Methods

The diagnosis methods of the present invention involve diagnosing thephenotype of a test ERα or the genotype of a polynucleotide encoding thetest ERα. In the genotype diagnosis methods, it can be determinedwhether the polynucleotide encoding the test ERα contains a valiantcodon therein which provides for the one or more substituted amino acidswhich confer the activity for transactivation of the reporter gene, asdescribed in the above 5.2. In the phenotype diagnosis methods, it canbe determined whether the test ERα contains one or more substitutedamino acids therein which confer the activity for transactivation of thereporter gene as described in the above 5.2.

The genotype diagnosis methods typically involve preparing the test ERαpolynucleotide, searching for the variant codon and determining themutation in the variant codon, if present. Examples of such genotypediagnosis methods include PCR amplification and nucleotide sequencingmethods, single strand conformation polymorphism (SSCP) methods,restriction fragment length polymorphism (RFLP) methods, hybridizationmethods and the like.

The test ERα polynucleotide can be prepared for the genotype diagnosismethods by preparing test genomic DNAs or test cDNA. In such cases, testgenomic DNAs or test cDNAs, which contain the test ERα polynucleotide,are collectively prepared from a test sample obtained from a testanimal, such as a test human. Such a test sample may be obtained fromnon-surgical methods, from surgical methods such as from a fine needleor from a biopsy or the like. Examples of such test samples include thecellular tissue of the test mammal, such as hair, peripheral blood, oralepithelial tissue, liver, prostate, ovaries, uterus, mammary gland orthe like, from which test genomic DNAs or test cDNAs can be extracted.

For example, the test genomic DNAs can be prepared according to themethods described in TAKARA PCR Technical news No. 2 (Takara Shuzo,1991.9). In such cases, a test sample of 2 to 3 hairs from a test mammalare washed with sterile water and ethanol and are cut into 2 to 3 mm inlength. The test cells in the hairs are then lysed with a sufficientamount, such as 200 μl, of BCL buffer (10 mM of Tris-HCl (pH 7.5), 5 mMof MgCl₂, 0.32 M of sucrose, 1% of Triton X-100). The test genomic DNAstherefrom are washed from unnecessary proteins by adding and mixingProteinase K and SDS to the lysed test cells to amount to finalconcentrations of 100 μl/ml and 0.5% (w/v), respectively. Afterincubating the reaction mixture at 70° C., the test genomic DNAs can bepurified by a phenol-chloroform extraction.

Additionally, when the test sample is peripheral blood, test genomicDNAs can be collectively obtained, for example, by processing the testsample with DNA-Extraction kit (Stratagene).

Also, when the test sample is obtained from a biopsy, the test cDNAs maybe prepared from the test sample by collectively reverse transcribingthe RNAs in the cellular tissue. The RNAs can be collectively obtainedfrom the cellular tissue by using TRIZOL reagent (Gibco), and preferablywhen the cellular tissue is still fresh.

Furthermore, the test genomic DNAs can be prepared according to themethods described in M. Muramatsu “Labo-Manual-Idenshi-Kogaku” (Maruzen,1988).

Even furthermore, the test cDNAs may be prepared according to thegenetic engineering techniques described in J. Sambrook, E. F. Frisch,T. Maniatis, “Molecular Cloning 2nd edition”, Cold Spring HarborLaboratory, 1989, as described in the above 5.2.

When searching for the variant codon in the genotype diagnosis methods,a searching region in the test ERα polynucleotide typically includescodons therein which are suspected to be the variant codon. As such, thesearching region in the test ERα polynucleotide may include the codonsin the test ERα polynucleotide which encode the amino acids in the testERα at relative positions 303 to 578. For example, such genotypediagnosis methods may have the searching region include a codon in thetest ERα polynucleotide which encode an amino acid at relative positionsselected from 303, 309, 390, 396, 415, 494, 531, 578 and the like.

The PCR amplification and sequencing methods as well as the SSCP methodsmay then use the prepared test cDNAs or the test genomic DNAs tospecifically PCR amplify the searching regions in the test ERαpolynucleotide therefrom. Search oligonucleotides can be utilized tospecifically PCR amplify from the test cDNAs or test genomic DNAs, thesearching regions present in the test ERα polynucleotide.

The search oligonucleotides in this PCR amplification are typicallydesigned to specifically PCR amplify the searching region in the testERα polynucleotide. The search oligonucleotides may have a size of from8 to 50 bp, preferably 15 to 40 bp, and may have a GC content of 30% to70%. Such search oligonucleotides may be synthesized with a DNAsynthesizer using the β-cyanoethyl phosphoamidide methods, thiophosphitemethods or the like. Further, the search oligonucleotides may beunlabeled, non-radioactively labeled, radiolabeled such as with ³²P orthe like. The PCR amplification typically utilizes a combination of aforward search oligonucleotide and a reverse search oligonucleotide tospecifically PCR amplify the searching region in the test ERαpolynucleotide. Examples of such combinations of forward and reversesearch oligonucleotides for a human test ERα polynucleotide are shownbelow in Table 3, in connection with the relative position of the aminoacid encoded in the searching region.

TABLE 3 SEQ IDs depicting the search oligonucleotides Reverse relativeForward search oligonucleotide search oligonucleotide position SEQ ID:29, SEQ ID: 30, SEQ SEQ ID: 34, SEQ ID: 35, 303 ID: 31, SEQ ID: 32 orSEQ ID: SEQ ID: 36, SEQ ID: 37 or 33 SEQ ID: 38 SEQ ID: 39, SEQ ID: 40,SEQ SEQ ID: 44, SEQ ID: 45, 309 ID: 41, SEQ ID: 42 or SEQ ID: SEQ ID:46, SEQ ID: 47 or 43 SEQ ID: 48 SEQ ID: 49, SEQ ID: 50, SEQ SEQ ID: 54,SEQ ID: 55, 390 ID: 51, SEQ ID: 52 or SEQ ID: SEQ ID: 56, SEQ ID: 57 or53 SEQ ID: 58 SEQ ID: 59, SEQ ID: 60, SEQ SEQ ID: 64, SEQ ID: 65, 396ID: 61, SEQ ID: 62 or SEQ ID: SEQ ID: 66, SEQ ID: 67 or 63 SEQ ID: 68SEQ ID: 69, SEQ ID: 70, SEQ SEQ ID: 74, SEQ ID: 75, 415 ID: 71, SEQ ID:72 or SEQ ID: SEQ ID: 76, SEQ ID: 77 or 73 SEQ ID: 78 SEQ ID: 79, SEQID: 80, SEQ SEQ ID: 84, SEQ ID: 85, 494 ID: 81, SEQ ID: 82 or SEQ ID:SEQ ID: 86, SEQ ID: 87 or 83 SEQ ID: 88 SEQ ID: 89, SEQ ID: 90, SEQ SEQID: 94, SEQ ID: 95, 531 ID: 91, SEQ ID: 92 or SEQ ID: SEQ ID: 96, SEQID: 97 or 93 SEQ ID: 98 SEQ ID: 99, SEQ ID: 100, SEQ SEQ ID: 104, SEQID: 105, 578 ID: 101, SEQ ID: 102 or SEQ SEQ ID: 106, SEQ ID: 107 or ID:103 SEQ ID: 108

The searching regions in the test ERα polynucleotide can be specificallyPCR amplified from the test cDNAs or the test genomic DNAs according tothe methods described in Saiki et al., Science, vol. 230, pp. 1350-1354(1985). The PCR mixture in this PCR amplification may contain 1.5 mM to3.0 mM magnesium chloride, heat tolerant DNA polymerase, dNTPs (dATP,dTTP, dGTP, and dCTP), one of the forward search oligonucleotides incombination with one of the reverse search oligonucleotides and the testgenomic DNAs or test cDNAs. In this PCR amplification, there may berepeated 20 to 50 times, preferably 25 to 40 times, an incubation cycleentailing a denaturation incubation, an annealing incubation and anelongation incubation. The denaturation incubation may incubate the PCRmixture at 90° C. to 95° C., and preferably at 94° C. to 95° C., for 1min to 5 min, and preferably for 1 min to 2 min. The annealingincubation following the denaturing incubation may incubate the PCRmixture at 30° C. to 70° C., and preferably at 40° C. to 60° C., for 3seconds to 3 minutes, and preferably for 5 seconds to 2 minutes. Theelongation incubation following the denaturing incubation may incubatethe PCR mixture at 70° C. to 75° C., and preferably at 72° C. to 73° C.,for about 15 seconds to 5 minutes, and preferably for 30 seconds to 4minutes.

When utilizing the PCR amplification and nucleotide sequencing methods,the genotype diagnosis methods may then entail subjecting the resultingPCR mixture to low melting point agarose gel electrophoresis. Theamplified polynucleotide encoding the searching region (hereinafterreferred to as searching region polynucleotide) is recovered from thelow melting point agarose gel and is sequenced to provide a nucleotidesequence of the searching region polynucleotide.

The mutation in the variant codon, if present, may then be determined bysequencing the searching region polynucleotide and by determining themutation in the nucleotide sequence. In sequencing the searching regionpolynucleotide, there may be utilized the direct sequencing methods oran automated sequencing method. Examples of the direct sequencingmethods include manual sequencing methods (Maxam Gilbert methoddescribed in Maxam, A. M. & W. Gilbert, Proc. Natl. Acad. Sci. USA, 74,560, 1977), the Sanger method (described in Sanger, F. & A. R. Coulson,J. Mol. Biol., 94, 441, 1975 as well as Sanger, F., Nicklen, and A. R.,Coulson, Proc. Natl. Acad. Sci. USA., 74, 5463, 1977), the methodsdescribed in BioTechniques, 7, 494 (1989) and the like. When anautomated DNA sequencer such as ABI autosequencer (Model 377. AppliedBiosystems) is used, an appropriate DNA sequencing kit such as ABI BigDye terminator cycle sequencing ready reaction kit can be used toprepared the searching region for the automated DNA sequencer. Aftersequencing, the nucleotide sequence of the searching regionpolynucleotide may then be compared to a nucleotide sequence encoding anormal ERα to determine the mutation in the valiant codon, if present,in the searching region.

When utilizing the SSCP methods, the resulting PCR mixture is subjectedto a native polyacrylamide gel electrophoresis according to the methodsdescribed in Hum. Mutation, vol. 2, p. 338. In such cases, it ispreferable that the PCR amplification above utilize the radiolabeledoligonucleotides so that the searching region polynucleotide isradiolabeled and the searching region polynucleotide can be detected inthe native polyacrylamide gel by employing the radioactivity thereof. Insuch SSCP methods, the radiolabeled searching region polynucleotide canbe heat-denatured into single strand polynucleotides and subjected tothe native polyacrylamide gel electrophoresis in a buffer to separateeach of the single strand polynucleotides. Examples of buffers which maybe utilized in the native polyacrylamide gel electrophoresis includeTris-phosphate (pH 7.5-8.0), Tris-acetate (pH 7.5-8.0), Tris-borate (pH7.5-8.3) and the like, with Tris-borate (pH 7.5-8.3) being preferred. Inaddition, auxiliary components for the native polyacrylamide gelelectrophoresis may be utilized in the buffer, such as EDTA. Theconditions for such native polyacrylamide gel electrophoresis mayinclude a constant power of 30 to 40 W at 4° C. to room temperature(about 20 to 25° C.) for 1 hour to 4 hours.

After the native polyacrylamide gel electrophoresis, the nativepolyacrylamide gel is transferred onto a filter paper and contacted withX-ray film to expose the X-ray film with the radiation from theradiolabeled searching region polynucleotide. An appropriate cassettemay be utilized to expose the X-ray film. The autoradiogram obtainedfrom developing the X-ray film allows a comparison of the mobility ofthe radiolabeled searching region polynucleotide with the mobility of astandard. Such a mobility of the standard can be the mobility expectedwhen the searching region polynucleotide is composed of only normalcodons of the normal ERα polynucleotide. A mobility of the radiolabeledsearching region polynucleotide different from the mobility of thestandard typically indicates that there is one or more valiant codons inthe radiolabeled searching region.

The radiolabeled searching region polynucleotide may then be recoveredfrom the native polyacrylamide gel by using heated or boiling water. Theradiolabeled searching region may be PCR amplified for a second roundand then prepared for sequencing. The mutation in the variant codon, ifpresent, may then be determined similarly to the methods described abovein the PCR amplification and nucleotide sequencing methods.

The hybridization methods typically utilize a probe oligonucleotide toobserve whether the probe oligonucleotide can hybridize to the searchingregions. The searching regions can be provided in the hybridizationmethods by utilizing the searching region polynucleotide, the preparedtest cDNA, the prepared test genomic DNA, a purified test ERαpolynucleotide or the like. Further, the hybridization methods mayrestriction digest the searching region polynucleotide and then utilizethe restriction digested searching region polynucleotide to observewhether the probe oligonucleotide can hybridize thereto.

The probe oligonucleotides may have a size of from 15 to 40 bp, and mayhave a GC content of 30% to 70%. Such probe oligonucleotides may besynthesized with a DNA synthesizer using the β-cyanoethyl phosphoamididemethods, thiophosphite methods or the like. Further, the probeoligonucleotides are typically non-radioactively labeled such as withbiotin, radiolabeled such as with ³²P or the like.

The probe oligonucleotides may be composed of the nucleotide sequence ofthe searching region, when the searching region is composed of onlynormal codons of a normal ERα polynucleotide. Such a nucleotide sequenceallows the probe oligonucleotides to hybridize to the searching regionin the test ERα polynucleotide under stringent conditions, when thesearching region therein is composed of only normal codons of a normalERα polynucleotide. Examples of such probe oligonucleotides for a humantest ERα polynucleotide are shown below in Table 3, in connection withthe relative position of the amino acid encoded in the searching region.

TABLE 3 Probe oligonucleotide relative position SEQ ID: 111, SEQ ID:112, SEQ 303 ID: 113, SEQ ID: 114 or SEQ ID: 115 SEQ ID: 116, SEQ ID:117, SEQ 309 ID: 118, SEQ ID: 119 or SEQ ID: 120 SEQ ID: 121, SEQ ID:122, SEQ 390 ID: 123, SEQ ID: 124 or SEQ ID: 125 SEQ ID: 126, SEQ ID:127, SEQ 396 ID: 128, SEQ ID: 129 or SEQ ID: 130 SEQ ID: 131, SEQ ID:132, SEQ 415 ID: 133, SEQ ID: 134 or SEQ ID: 135 SEQ ID: 136, SEQ ID:137, SEQ 494 ID: 138, SEQ ID: 139 or SEQ ID: 140 SEQ ID: 141, SEQ ID:142, SEQ 531 ID: 143, SEQ ID: 144 or SEQ ID: 145 SEQ ID: 146, SEQ ID:147, SEQ 578 ID: 148, SEQ ID: 149 or SEQ ID: 150

Typically, the hybridization methods are conducted under stringentconditions. As such stringent conditions, for example, theprehybridization or hybridization treatments are conducted inprehybridization buffer and hybridization buffer, and the washings areconducted twice for 15 minutes in washing buffer. The hybridizationmethods may optionally have another washing for 30 minutes in a buffercontaining 0.1×SSC (0.015M NaCl, 0.0015M sodium citrate) and 0.5% SDS.As the prehybridization buffer, there may be utilized a buffercontaining 6×SSC (0.9M NaCl, 0.09M sodium citrate), 5×Denhart (0.1%(w/v) phycol 400, 0.1% (w/v) polypyrolidone and 0.1% BSA), 0.5% (w/v)SDS and 100 μg/ml of salmon sperm DNA. Also as the prehybridizationbuffer, there may be utilized a DIG EASY Hyb buffer (Boehringer Manheim)to which salmon sperm DNA is added to a concentration of 100 μg/ml.Further, as the prehybridization buffer, there may be utilized a buffercontaining 6×SSPE (0.9M NaCl, 0.052M NaH₂PO₄, 7.5 mM EDTA), 0.5% SDS,5×Denhart and 0.1 mg/ml of salmon sperm DNA. As the hybridizationbuffer, there may be utilized the prehybridization buffer to which theprobe oligonucleotide is added to a sufficient amount. The temperatureof the prehybridization and hybridization treatments can vary with thelength of the probe oligonucleotide and for example, may be at the Tmvalue of the probe oligonucleotide to a temperature that is 2 to 3 lowerthan the Tm value of the probe oligonucleotide. The temperature of thewashings can also vary with the length of the oligonucleotide, and forexample may be conducted at room temperature. The Tm value in suchcases, can be achieved by estimating the quantity of nucleotide unitsthat should form hydrogen bonds in the hybridization buffer with thenucleotide units in the probe oligonucleotide, and then by adding thetemperatures achieved from adding 2° C. for the A or T nucleotide unitsin the probe oligonucleotide which should form the hydrogen bond andadding 4° C. for the G or T nucleotide units in the probeoligonucleotide which should form the hydrogen bond.

For example, the hybridization methods can involve dot-blothybridization methods, mismatch detection methods or the like.

The dot-blot hybridization methods typically involve fixing the test ERαpolynucleotide to a membrane and evaluating whether the probeoligonucleotide can hybridize to the searching region in the fixed testERα polynucleotide. In fixing test ERα polynucleotide onto the membrane,there can be utilized as the test ERα polynucleotide, the searchingregion polynucleotide, the prepared test cDNA, the prepared test genomicDNA, a purified test ERα polynucleotide or the like. The test ERαpolynucleotide can be fixed to the membrane by incubating the test ERαpolynucleotide at 90 to 100° C. for 3 to 5 min, by spotting the test ERαpolynucleotide onto the membrane, by drying the resulting membrane andby exposing the spotted searching region with UV light. As the membrane,there can be utilized a nylon membrane such as Hybond N (AmerschamPharmacia). The probe oligonucleotide can then be utilized to, evaluatewhether the probe oligonucleotide can hybridize to the searching region.The probe oligonucleotide may be utilized by incubating the probeoligonucleotide and the test ERα polynucleotide at 40 to 50° C. for 10to 20 hours. The resulting membrane may then be washed and thehybridized probe oligonucleotide can then be detected, if present.

When the probe oligonucleotide is radiolabeled with ³²P, the hybridizedprobe oligonucleotide, if present, may be detected by exposing theresulting membrane to a X-ray film.

When the probe oligonucleotide is nonradioactively labeled with biotin,the hybridized probe oligonucleotide, if present, may be detected with aspacer and a hybridization detection enzyme such as biotinylatedalkaline phosphatase, biotinylated peroxidase or the like. When theprobe oligonucleotide labeled with biotin can hybridize to the searchingregion, the spacer, such as streptavidin, can bind to the hybridizedprobe oligonucleotide labeled with biotin such that the hybridizationdetection enzyme can then connect to the hybridized probeoligonucleotide labeled with biotin through the spacer. The connectedhybridization detection enzyme can then participate in a reaction toindicate whether the probe oligonucleotide has hybridized to thesearching region in the test ERα polynucleotide. The enzymatic reactioncan provide a change in color or a luminescence.

When the probe oligonucleotide does not hybridize to the searchingregion, it can be determined that the searching region contains one ormore of the variant codons. The searching region may then be sequenced.The mutation in the variant codon, if present, may be determinedsimilarly to the methods described above in the PCR amplification andnucleotide sequencing methods.

The mismatch detection methods are described in Biswas, I. and Hsieh,P., J. Biol. Chem., 271(9), pp. 5040-5048 (1996) as well as Nippon geneinformation, 1999, No. 125, Nippon Gene, Toyama. In such mismatchdetection methods, a mismatch detection enzyme, such as Taq Mut S, isutilized to search certain mismatches in the hybridization of the probeoligonucleotide to the searching region. The mismatch detection enzymeallows the mismatches in the hybridization of the probe oligonucleotidewith the searching region to be detected at high temperatures such as ata temperature of 75° C. or lower. Typically, such mismatches in thehybridization thereof, bound with the mismatch detection enzyme, can bedetected by a gel shift assay or by the dot blot hybridization methodsas described in the above. When the mismatch detection enzyme can bindto a mismatched hybridization of the probe oligonucleotide and thesearching region, it may be determined that the searching regioncontains one or more of the valiant codons. The searching region may besequenced. The mutation in the variant codon, if present, may then bedetermined similarly to the methods described above in the PCRamplification and nucleotide sequencing methods.

Further, in the RFLP methods, a restriction enzyme is mixed with thetest ERα polynucleotide under reacting conditions. Typically, therestriction enzyme has a restriction site overlapping with the codon inthe searching region, which is suspected to be the variant codon. Asuccessful or an unsuccessful restriction digest at the restriction sitecan determine whether there is the variant codon in the searchingregion. The results of the restriction digestion can be evaluated by gelelectrophoresis analysis, such as with low melting point agarose gelelectrophoresis. The searching region may then be sequenced, if needed.The mutation in the variant codon, if present, may then be determinedsimilarly to the methods described above in the PCR amplification andnucleotide sequencing methods.

The phenotype diagnosis methods may involve searching in an amino acidsequence of the test ERα for one or more substituted amino acids whichconfer the activity for transactivation of the reporter gene asdescribed in the above 5.2. After searching for the substituted aminoacid, the mutation in the test ERα, if present, is determined bycomparing the amino acid sequence of the test ERα to the amino acidsequence of the normal ERα. To search for the substituted amino acid inthe test ERα, the searching region in the test ERα may include the aminoacids in the test ERα at relative positions 303 to 578. For example,such phenotype diagnosis methods may have the searching region includean amino acid in the test ERα at one or more relative positions selectedfrom 303, 309, 390, 396, 415, 494, 531, 578 and the like.

To search for the substituted amino acid in the test ERα, an antibodyhaving an epitope in the searching region in the test ERα may be useful.A successful or unsuccessful binding of such an antibody can determinewhether there is a substituted amino acid at the searching region in thetest ERα. The mutation in the test ERα can then be determined bycomparing the amino acid sequence of the test ERα with the amino acidsequence of a normal ERα.

The test ERα may be prepared from a test sample by cell extracttechniques. Further, the test ERα may be prepared for the phenotypediagnosis methods by purifying recombinant test ERα.

5.4. The Reporter Assay with the Test ERα

A test ERα can be assayed for the activity for transactivation of thereporter gene, described in the above 5.2., by utilizing an assay cellcomprising the test ERα and a chromosome which comprises the reportergene. In such cases, the assay cell is typically exposed to a ligand andthe transactivation level of the reporter gene is measured toquantitatively analyze the activity for transactivation of the reportergene by the test ERα. Further, the activity for transactivation of thereporter gene by the test ERα can be evaluated by comparing thetransactivation level of the reporter gene by the test ERα to thetransactivation level of the reporter gene by a standard. Furthermore,the test ERα can be screened by selecting the test ERα in which thetransactivation level of the reporter gene by the test ERα is differentthan the transactivation level by the standard.

The assay cell can be produced by introducing the reporter gene and agene encoding the test ERα into a host cell. The reporter gene isinserted into a chromosome of the host cell. The test ERα gene can beintroduced into the host cell for transient expression or can beintroduced into the host cell so that the test ERα gene is inserted intoa chromosome of the host cell. When inserting the test ERα gene into achromosome of the host cell, the test ERα gene may be inserted into thechromosome together with the test ERα or into another chromosome in thehost cell. Additionally, the reporter gene may be introduced into thehost cell to produce a stably transformed cassette cell, as described inthe above 5.2., and the test ERα gene may then be introduced into thestably transformed cassette cell, as described in the above 5.2.

The test ERα gene is introduced into the host cell so that the test ERαgene can be expressed in the assay cell to provide the test ERα. In thisregard, such a test ERα gene typically comprises a promoter linkedoperably upstream from a polynucleotide which encodes the test ERα.

To introduce the test ERα gene into the host cell, conventionaltechniques for introducing the test ERα gene may be applied according tothe type of host cell, as described in the above 5.2. In this regard,when test ERα is introduced into the host cell for transient expression,the test ERα gene is introduced in a circular form. When inserting thetest ERα gene into the chromosome of the host cell, the test ERα isintroduced in a linearized form. Also, a vector may be utilized tointroduce the test ERα gene or the reporter gene into the host cell, asdescribed in the above 5.2.

Further, the test ERα gene can be introduced into the stably transformedcassette cell to provide the assay cell. In such cases, the test ERαgene may also be introduced into the stably transformed cassette cell toprovide a stably transformed binary cell. Such an stably transformedbinary cell has the chromosomes thereof stably comprise the test ERαgene and the reporter gene.

The host cell utilized to produce the assay cell typically fails have anexpressed normal or mutant ERα. Examples of the host cells includeeukaryotic cells such as HeLa cells, CV-1 cells, Hepa1 cells, NIH3T3cells, HepG2 cells, COS1 cells, BF-2 cells, CHH-1 cells and the like.

In the reporter assay, the assay cell is typically exposed with asufficient amount of a ligand for one to several days. Further, theligand can be exposed to the assay cell under agonistic conditions orantagonistic conditions directed to the test ERα. The agonisticconditions typically have the assay cell exposed to the ligand as thesole agent probable of stimulating the test ERα. The antagonisticconditions typically have the assay cell exposed to the ligand and E2.

As the ligand there is usually utilized a ligand that is purely orpartially antagonistic or agonistic to the normal ERα. Examples of suchligands include the partial anti-estrogens such as tamoxifen,4-hydroxytamoxifen and raloxifene, the pure anti-estrogens such as ICI182780 (Wakeling A E et al., Cancer Res., 512:3867-3873 (1991)) and ZM189154 (Dukes M et al., J. Endocrinol., 141:335-341 (1994)) and thelike.

After exposure, the transactivation level of the reporter gene ismeasured by measuring the expression level of the reporter gene. In suchcases, the reporter protein or the reporter RNA (encoded by the reportersequence) is stored in the cell or is secreted from the cell so that theexpression level can be measured therewith. The expression level of thereporter gene can be measured by a Northern blot analysis, by a Westernblot analysis or by measuring the activity level of the reporterprotein. The activity level of the protein typically indicates the levelat which the reporter protein is expressed.

For example, when the reporter gene encodes luciferase as the reporterprotein, the expression level of the reporter gene can be measured bythe luminescence provided by reacting luciferin and luciferase. In suchcases, a crude cell extract is produced from the cells and luciferin isadded to the crude cell extract. The luciferin may be allowed to reactwith the luciferase in the cell extract at room temperature. Theluminescence from adding luciferin is usually measured as an indicatorof the expression level of the reporter gene, since the crude cellextract produces a luminescence at a strength proportional to the levelof luciferase expressed in the cell and present in the crude cellextract. A luminometer may be utilized to measure the luminescence inthe resulting crude cell extract.

The measured transactivation level can then be compared with atransactivation level of the reporter gene by a standard to evaluate theactivity for transactivation by the test ERα. Such a transactivationlevel of the reporter gene by the standard in evaluating the activityfor transactivation by the test ERα can be the expected transactivationlevel of the reporter gene in cases in which the assay cell expressesthe normal ERα or an ERα which phenotype is known (instead of the testERα). When the measured transactivation level provided by the test ERαis different than the transactivation level of the reporter gene by thestandard, the test ERα may be selected as a mutant ERα.

Furthermore, mutant ligand dependent transcription factors can bescreened. In such cases, the a gene encoding the test ligand dependenttranscription factor, instead of the test ERα gene, in introduced intothe host cell. Examples of such test ligand dependent transcriptionfactors include a test ERβ, a test AR, a test GR, a test TR, a test PR,a test PXR, a test lipophilic vitamin receptor such a test VDR and atest RAR and the like. The reporter gene in such cases comprises anappropriate receptor responsive sequence cognate with the provided testligand dependent transcriptional factor, instead of the ERE.

6. EXAMPLES 6.1. Example 1 A Polynucleotide Encoding the Human MutantERα 6.1.1. Production of a Plasmid Encoding Human Normal ERα

A human liver cDNA library (CLONETECH, Quick clone cDNA#7113-1) isutilized to specifically PCR amplify therefrom a cDNA encoding a humannormal ERα. The PCR mixture in this PCR amplification contains 10 ng ofthe human liver cDNA library, 10 pmol of an oligonucleotide depicted inSEQ ID:11, 10 pmol of a oligonucleotide depicted in SEQ ID:12, LA-TaqPolymerase (Takara Shuzo), the buffer provided with the LA-TaqPolymerase and dNTPs (dATP, dTTP, dGTP, dCTP). The oligonucleotidesdepicted in SEQ ID:11 and SEQ ID:12 are synthesized with a DNAsynthesizer (Model 394, Applied Biosystems). In this PCR amplification,there is repeated 35 times with a PCRsystem 9700 (Applied Biosystems),an incubation cycle entailing an incubation at 95° C. for 1 minutefollowed by an incubation at 68° C. for 3 minutes.

The resulting PCR mixture is subjected to low melting point agarose gelelectrophoresis (Agarose L: Nippon Gene) to confirm that the amplifiedcDNA from the PCR amplification has a size of about 1.8 kb. Afterrecovering the amplified cDNA from the low melting point agarose gel, asample of the recovered cDNA is prepared with a Dye Terminator SequenceKit FS (Applied Biosystems). The prepared sample of the cDNA issequenced with an ABI autosequencer (Model 377, Applied Biosystems), toreveal that the cDNA has a nucleotide sequence encoding a human normalERα which has the amino acid sequence shown in SEQ ID:1.

Another PCR amplification is then similarly conducted to add a Kozakconsensus sequence immediately upstream from the start codon (ATG) inthe cDNA. In this PCR amplification, there is utilized 100 ng of thecDNA, a oligonucleotide depicted in SEQ ID:151 and an oligonucleotidedepicted in SEQ ID:12. The resulting PCR mixture is subjected to lowmelting point agarose gel electrophoresis (Agarose L: Nippon Gene) toconfirm that the amplified cDNA from the PCR amplification has a size ofabout 1.8 kb. After recovering the amplified cDNA from the low meltingpoint agarose gel, 1 μg of the amplified cDNA is treated with a DNABlunting Kit (Takara Shuzo) to blunt the ends of the amplified cDNA.Subsequently, the resulting cDNA therefrom is allowed to react with a T4polynucleotide kinase to phosphorylate the ends thereof. After phenoltreating the phosphorylated cDNA, the phosphorylated cDNA is ethanolprecipitated to achieve a purified form of the phosphorylated cDNA.

The plasmid pRc/RSV (Invitrogen) is restriction digested withrestriction enzyme Hind III and is then treated with bacterial alkalinephosphatase (BAP) for 1 hour at 65° C. The restriction digested pRc/RSVis then purified by a phenol treatment and ethanol precipitation. Therestriction digested pRc/RSV is treated with a DNA Blunting Kit (TakaraShuzo) to blunt the ends thereof and is subjected to low melting pointagarose gel electrophoresis (Agarose L, Nippon Gene). After recoveringthe restriction digested pRc/RSV from the low melting point agarose gel,100 ng of the restriction digested pRc/RSV and all of the above purifiedform of the phosphorylated cDNA are used in a ligation reaction with aT4 DNA ligase. The ligation reaction mixture is used to transform E.coli competent DH5α cells (TOYOBO). The transformed E. coli cells arecultured in LB (Luria-Bertani) medium to which ampicillin is added to aconcentration of 50 μg/ml (hereinafter referred to as LB-amp medium; J.Sambrook, E. F. Frisch, T. Maniatis; Molecular Cloning 2nd Edition, ColdSprings Harbor Laboratory Publishing, 1989). The clones thereof showingan ampicillin resistance are then recovered. Some of the clones are thenused to isolate therefrom the plasmids derived from the ligationreaction. An aliquot sample of each of the isolated plasmids are thenprepared with a Dye Terminator Sequence Kit FS (Applied Biosystems). Theprepared plasmids are sequenced with an ABI autosequencer (Model 377,Applied Biosystems), to confirm that there is a plasmid that has anucleotide sequence encoding human normal ERα having the amino acidsequence shown in SEQ ID:1. Such a plasmid is selected and is designatedas pRc/RSV-hERαKozak.

6.1.2. Production of Plasmids Encoding the Human Mutant ERα K303R,S309F, M396V, G415V, G494V or K531E

6.1.2.1. Production of a Plasmid for Mutagenesis

The plasmid pRc/RSV-hERαKozak is restriction digested with restrictionenzyme Not I for 1 hour at 37° C. The restriction digestion reactionmixture is subjected to low melting point agarose gel electrophoresis(Agarose L: Nippon Gene) to confirm that there is DNA fragment having asize of about 1.6 kb. The 1.6 kb DNA fragment is then recovered from thelow melting point agarose gel.

The plasmid pBluescriptII SK(+) (Stratagene) is restriction digestedwith NotI for 1 hour at 37° C. and is then treated with BAP for 1 hourat 65° C. The restriction digestion reaction mixture is subjected to lowmelting point agarose gel electrophoresis (Agarose L: Nippon Gene) andthe restriction digested pBluescriptII SK(+) is recovered from the lowmelting point agarose gel. Subsequently, 100 ng of the above 1.6 kb DNA,fragment and 100 ng of the recovered pBluescriptII SK(+) are used in aligation reaction with T4 DNA ligase. The ligation reaction mixture isused to transform E. coli competent DH5α cells (TOYOBO). The transformedE. coli cells are cultured in LB-amp medium. The clones thereof showingan ampicillin resistance are then recovered. Some of the clones are thenused to isolate therefrom the plasmids derived from the ligationreaction. An aliquot sample of each of the isolated plasmids are thenrestriction digested with restriction enzymes Not I and Hind III. Therestriction digestion reaction mixtures are subjected to agarose gelelectrophoresis. It is then confirmed that there is plasmid in which theplus strand in the plasmid contains the sense strand encoding the humannormal ERα operably with M13 microphage replication origin (fl ori). Inthis regard, it is confirmed that there is a plasmid which has astructure such that when the fl ori replicates one of the strands in theplasmid, the sense strand encoding human normal ERα would be replicatedtherewith. Such a plasmid is selected and is designated as pSK-NN.

6.1.2.2. Site Directed Mutagenesis at Relative Positions 303, 309, 396,415, 494 and 531

According to the methods described in McClary J A et al. (Biotechniques1989(3): 282-289), specified mutations are introduced into thepolynucleotide encoding the human normal ERα. Such procedures aredescribed in relation with the present invention below.

The plasmid pSK-NN, provided in the above 6.1.2.1., is utilized totransform E. coli competent CJ236 cells (Takara Shuzo) according to theprotocol provided with the E. coli competent CJ236 cells. A clonethereof showing ampicillin resistance is then cultured for 16 hours in aLB-amp medium. Subsequently, a colony of the clone is suspended in 10 mlof a 2×YT medium to which a M13 helper phage is added to a concentrationof at least 1×10¹¹ pfu/ml (hereinafter referred to as 2×YT-M13) medium.After culturing the clone in the 2×YT-M13 medium for 2 hours at 37° C.,kanamycin is added thereto to a concentration of 50 μg/ml and the cloneis then cultured for 22 hours. The resulting suspension is centrifugedand 8 ml of the resulting supernatant is transferred to a 15 ml testtube. Two milliliters (2 ml) of 2.5M NaCl-40% PEG8000 (Sigma) is thenadded to and stirred with the supernatant. The supernatant isrefrigerated at 4° C. for 1 hour and is centrifuged (3,000 rpm, 2,000×g,10 minutes, 4° C.) to collect the phage therefrom as a pellet. After thephage is suspended in 400 μl of distilled water, an identical amount byvolume of phenol is added thereto and the resulting suspension is gentlyshook for 5 minutes. The resulting suspension is centrifuged so that theaqueous layer therein is extracted therefrom. For a second round ofphenol treatment, an identical amount by volume of phenol is then addedto the aqueous layer and is vigorously shook. The resulting suspensionis centrifuged so that the aqueous layer is extracted therefrom. To theaqueous layer from the second phenol treatment, an identical amount byvolume of chloroform is added thereto and is vigorously shook. Theresulting suspension is centrifuged (15,000 rpm, 20,000×g, 5 minutes, 4°C.) to extract the aqueous layer therefrom. To the aqueous layer fromthe chloroform treatment, there is added 800 μl of 100% ethanol and 50μl of 3M sodium acetate. After refrigerating the aqueous layer therefromat −80° C. for 20 minutes, the aqueous layer is centrifuged. Theresulting pellet therefrom is rinsed with 70% ethanol and is then dried.After pellet the residue in sterile water, the light absorbance ofaqueous solution is measured at a wavelength of 260 nm to calculate theamount of the single strand sense DNA encoding human normal ERα therein.

The oligonucleotides for the site directed mutagenesis are synthesizedto provide the oligonucleotides depicted in SEQ ID:152, SEQ ID:153, SEQID:154, SEQ ID:155, SEQ ID:156 and SEQ ID:157.

In using the oligonucleotide depicted in SEQ ID:152, the AAG codonencoding the lysine present at relative position 303 is changed to anAGG codon encoding arginine.

In using the oligonucleotide depicted in SEQ ID:153, the TCC codonencoding the serine present at relative position 309 is changed to a TTCcodon encoding phenylalanine.

In using the oligonucleotide depicted in SEQ ID:154, the ATG codonencoding the methionine present at relative position 396 is changed toan GTG codon encoding valine.

In using the oligonucleotide depicted in SEQ ID:155, the GGA codonencoding the glycine present at relative position 415 is changed to aGTA codon encoding valine.

In using the oligonucleotide depicted in SEQ ID:156, the GGC codonencoding the glycine present at relative position 494 is changed to aGTC codon encoding valine.

In using the oligonucleotide depicted in SEQ ID:157, the AAG codonencoding the lysine present at relative position 531 is changed to a GAGcodon encoding glutamic acid.

Each of the oligonucleotides is phosphorylated with 10 pmol of apolynucleotide kinase (Takara Shuzo) in the buffer provided with thepolynucleotide kinase. In the phosphorylation reactions, 2 mM of ATP isused in each of the reaction mixtures and the reaction mixtures areincubated at 37° C. for 30 minutes. Subsequently, about 1 pmol of thephosphorylated oligonucleotides are mixed, respectively, with 0.2 pmolof the single stand sense DNA encoding normal ERα. To produce 10 μlannealing reaction mixtures, the mixtures are then added, respectively,to annealing buffer (20 mM of Tris-Cl (pH7.4), 2 mM of MgCl₂, 50 mM ofNaCl). The annealing reaction mixtures are subjected to an incubation at70° C. for 10 minutes, then an incubation at 37° C. for 60 minutes,which is followed by an incubation at 4° C. Synthesizing reactionmixtures are then produced therefrom by adding, respectively, to theannealing reaction mixtures, 2 units (0.25 μl) of T7 DNA polymerase (NewEngland Labs), 2 units of (0.25 μl) of T4 DNA ligase (Takara Shuzo) and1.2 μl of a synthesizing buffer (175 mM of (Tris-Cl (pH 7.4), 375 mM ofMgCl₂, 5 mM of DTT, 4 mM of dATP, 4 mM of dCTP, 4 mM of dGTP, 4 mM ofdTTP and 7.5 mM of ATP). The synthesizing reaction mixtures areincubated at 4° C. for 5 minutes, incubated at room temperature for 5minutes, and then incubated at 37° C. for 2 hours, to providesynthesized DNA plasmids.

Two microliters (2 μl) of each of the synthesizing reaction mixtures arethen used to transform E. coli competent DH5α cells (TOYOBO). Thetransformed E. coli cells are cultured in LB-amp. The clones thereofshowing an ampicillin resistance are then recovered. Some of the clonesare then used to isolate therefrom the plasmids from the synthesizingreactions. An aliquot sample of each of the isolated plasmids are thenprepared with a Dye Terminator Sequence Kit FS (Applied Biosystems). Theisolated plasmids are sequenced with an ABI autosequencer (Model 377,Applied Biosystems).

It is confirmed from the sequencing that the isolated plasmidssynthesized from utilizing the oligonucleotide depicted in SEQ ID:152provides an isolated plasmid which has in the nucleotide sequenceencoding the human mutant ERα, an AGG codon corresponding to relativeposition 303, to provide arginine. Such an isolated plasmid is selectedand is designated as pSK-NN303.

It is confirmed from the sequencing that the isolated plasmidssynthesized from the oligonucleotide depicted in SEQ ID:153 provides anisolated plasmid which has in the nucleotide sequence encoding the humanmutant ERα, a TTC codon corresponding to relative position 309, toprovide phenylalanine. Such an isolated plasmid is selected and isdesignated as pSK-NN309.

It is confirmed from the sequencing that the isolated plasmidssynthesized from the oligonucleotide depicted in SEQ ID:154 provides anisolated plasmid which has in the nucleotide sequence encoding the humanmutant ERα, a GTG codon corresponding to relative position 396, toprovide valine. Such an isolated plasmid is selected and is designatedas pSK-NN396.

It is confirmed from the sequencing that the isolated plasmidssynthesized from the oligonucleotide depicted in SEQ ID:155 provides anisolated plasmid which has in the nucleotide sequence encoding the humanmutant ERα, a GTA codon corresponding to relative position 415, toprovide valine. Such an isolated plasmid is selected and is designatedas pSK-NN415.

It is confirmed from the sequencing that the isolated plasmidssynthesized from the oligonucleotide depicted in SEQ ID:156 provides anisolated plasmid which has in the nucleotide sequence encoding the humanmutant ERα, a GTC codon corresponding to relative position 494, toprovide valine. Such an isolated plasmid is selected and is designatedas pSK-NN494.

It is confirmed from the sequencing that the isolated plasmidssynthesized from the oligonucleotide depicted in SEQ ID:157 provides anisolated plasmid which has in the nucleotide sequence encoding the humanmutant ERα, a GAG codon corresponding to relative position 531, toprovide glutamic acid. Such as isolated plasmid is selected and isdesignated as pSK-NN531.

Table 4 below shows the utilized oligonucleotide for the mutagenesis,the produced plasmid therefrom and the resulting human mutant ERαtherefrom.

TABLE 1 SEQ ID of utilized oligonucleotide produced plasmid human mutantERα SEQ ID: 152 pSK-NN303 human mutant ERαK303R SEQ ID: 153 pSK-NN309human mutant ERαS309F SEQ ID: 154 pSK-NN396 human mutant ERαM396V SEQID: 155 pSK-NN415 human mutant ERαG415V SEQ ID: 156 pSK-NN494 humanmutant ERαG494V SEQ ID: 157 pSK-NN531 human mutant ERαK531E

The plasmids pSK-NN303, pSK-NN309, pSK-NN396, pSK-NN415, pSK-NN494 andpSK-NN531 are each restriction digested with restriction enzyme Not I at37° C. for 1 hour. Each of the restriction digestion reaction mixturesare then subjected to low melting point agarose gel electrophoresis toconfirm that there are DNA fragments having a size of about 1.6 kb. The1.6 kb DNA fragments are then recovered from the low melting pointagarose gel.

The plasmid pRc/RSV-hERαKozak, provided in 6.1.1., is restrictiondigested with restriction enzyme Not I at 37° C. for 1 hour and istreated with BAP at 65° C. for 1 hour. The restriction digestionreaction mixture is then subjected to low melting point agarose gelelectrophoresis to confirm that there is a DNA fragment having a size ofabout 5.5 kb. The 5.5 kb DNA fragment is then recovered from the lowmelting point agarose gel.

Subsequently, 100 ng of the recovered 5.5 kb DNA fragments are mixed,respectively, with 100 ng of the above 1.6 kb DNA fragments for aligation reaction with T4 DNA ligase. The ligation reaction mixtures areused to transform E. coli competent DH5α cells (TOYOBO). The transformedE. coli cells are cultured in LB-amp medium. The clones thereof showingan ampicillin resistance are then recovered. Some of the clones are thenused to isolate therefrom the plasmids derived from the ligationreactions. An aliquot sample of each of the isolated plasmids are thenrestriction digested with either restriction enzyme Not I or Mlu I. Therestriction digestion reaction mixtures are then subjected to agarosegel electrophoresis. It is confirmed that there are isolated plasmidswhich result from the each of the restriction digestions, DNA fragmentshaving the desired sizes. Such isolated plasmids provide DNA fragmentshaving sizes of 5.5 and 1.6 kb in the restriction digestions withrestriction enzyme Not I and provide DNA fragments of 7.1 kb inrestriction digestions with restriction enzyme Mlu I.

Each of the plasmids above is then PCR amplified with oligonucleotidesdepicted in SEQ ID:158, SEQ ID:159 and SEQ ID:160. The PCR mixtures inthese PCR amplifications contain one of the plasmids, theoligonucleotide depicted in SEQ ID:158, the oligonucleotide depicted inSEQ ID:159, the oligonucleotide depicted in SEQ ID:160, 400 μM of dNTPs(100 μM of dATP, 100 μM of dTTP, 100 μM of dGTP and 100 μM of dCTP),recombinant Taq DNA polymerase (Takara Shuzo), the PCR buffer providedwith the recombinant Taq DNA polymerase. In these PCR amplifications,there are repeated 30 times, an incubation cycle entailing an incubationat 94° C. for 30 seconds, then an incubation at 65° C. for 1 minute,which is followed by an incubation at 72° C. for 1 minute and 45seconds. Ten microliters (10 μl) of each of the resulting 25 μl PCRmixtures are subjected to a 1% agarose gel electrophoresis (Agarose S,Nippon Gene) to confirm that the resulting plasmids have a size of about1.2 kb. The plasmids are then prepared with a Dye Terminator SequenceKit FS (Applied Biosystems). The prepared samples of the plasmids aresequenced, respectively, with an ABI autosequencer (Model 377, AppliedBiosystems).

It is confirmed from the sequencing that the plasmid derived frompSK-NN303 encodes the human mutant ERα K303R (AAG→AGG; lysine→arginine;relative position 303). This plasmid is designated as pRc/RSV-hERαK303RKozak.

It is confirmed from the sequencing that the plasmid derived frompSK-NN309 encodes the human mutant ERα S309F (TCC→TTC;serine→phenylalanine; relative position 309). This plasmid is designatedas pRc/RSV-hERαS309F Kozak.

It is confirmed from the sequencing that the plasmid derived frompSK-NN396 encodes the human mutant ERα M396V (ATG→GTG;methionine→valine; relative position 396). This plasmid is designated aspRc/RSV-hERαM396V Kozak.

It is confirmed from the sequencing that the plasmid derived frompSK-NN415 encodes the human mutant ERα G415V (GGA→GTA; glycine→valine;relative position 415). This plasmid is designated as pRc/RSV-hERαG415VKozak.

It is confirmed from the sequencing that the plasmid derived frompSK-NN494 encodes the human mutant ERα G494V (GGC→GTC; glycine→valine;relative position 494). This plasmid is designated as pRc/RSV-hERαG494VKozak.

It is confirmed from the sequencing that the plasmid derived frompSK-NN531 encodes the human mutant ERα K531E (AAG→GAG; lysine→glutamicacid; relative position 531). This plasmid is designated aspRc/RSV-hERαK531E Kozak.

Table 5 below shows the plasmid utilized to produce the plasmid and theresulting plasmid produced therefrom.

TABLE 5 encoded plasmid produced plasmid human mutant ERα pSK-NN303pRc/RSV-hERK303R Kozak human mutant ERαK303R pSK-NN309 pRc/RSV-hERS309FKozak human mutant ERαS309F pSK-NN396 pRc/RSV-hERM396V Kozak humanmutant ERαM396V pSK-NN415 pRc/RSV-hERG415V Kozak human mutant ERαG415VpSK-NN494 pRc/RSV-hERG494V Kozak human mutant ERαG494V pSK-NN531pRc/RSV-hERK531E Kozak human mutant ERαK531E

6.1.3. Production of Plasmids Encoding the Human Mutant ERαG390D, S578Por G390D/S578P

6.1.3.1. Production of Plasmids Encoding the Human Mutant ERαG390D andS578P

The QuickChange Site-Directed Mutagenesis Kit (Stratagene) is used tomutagenize the plasmid pRc/RSV-hERα Kozak, described in the above6.1.1., so that the mutagenized plasmid encodes the human mutantERαG390D or the human mutant ERαS578P. In using the oligonucleotidesdepicted in SEQ ID: 17 and SEQ ID: 18, the GGT codon encoding theglycine present at relative position 390 is changed to a GAT variantcodon encoding aspartic acid. In using the oligonucleotides depicted inSEQ ID:27 and SEQ ID:28, the TCC codon encoding the serine present atrelative position 578 is changed to a CCC variant codon encodingproline. The manual provided with the QuickChange Site-DirectedMutagenesis Kit is used to produce the plasmids pRc/RSV-hERαG390D Kozak(GGT→GAT; glycine→aspartic acid; relative position 390) andpRc/RSV-hERαS578P Kozak (TCC→CCC; serine→proline; relative position578). The plasmids pRc/RSV-hERαG390D Kozak and pRc/RSV-hERαS578P Kozakare sequenced to confirm that the plasmids encoding the human mutant ERαcontain the desired mutation therein at relative position 390 or 578.

The QuickChange Site directed Mutagenesis Kit (Stratagene) is then usedto mutagenize pRc/RSV-hERαG390D Kozak so that the mutagenized plasmidencodes the human mutant ERαG390D/S578P. The oligonucleotides depictedin SEQ ID:27 and SEQ ID:28 are used to produce plasmidpRc/RSV-hERαG390D/S578P Kozak (GGT→GAT; glycine→aspartic acid; relativeposition 390 and TCC→CCC; serine→proline; relative position 578). Theplasmid pRc/RSV-hERαG390D/S578P Kozak is sequenced to confirm that theplasmid encoding the human mutant ERα contains the desired mutationstherein at relative positions 390 and 578.

6.1.3.2. Preparation from a Test Human Liver Tissue Sample of a PlasmidEncoding a Human Mutant ERαG390D/S578P

A frozen sample of test human liver tissue was utilized to obtain apolynucleotide encoding a human mutant ERαG390D/S578P. In utilizing thetest human liver tissue sample, 0.1 g of the test human liver tissuesample was homogenized with a homogenizer in 5 ml of a buffer containing4M guanidium thiocyanate, 0.1M Tris-HCl (pH 7.5) and 1% βmercaptoethanol. The resulting buffer was layered with 25 ml of anaqueous 5.7M CsCl solution and was ultracentrifuged at 90,000×g for 24hours to obtain a RNA pellet. After rinsing the RNA pellet with 70%ethanol, the RNA pellet was allowed to dry at room temperature. The RNApellet was then dissolved in 10 μl of sterile water to a concentrationof 1.2 μg/ml. Test cDNAs were then produced by collectively using theRNAs in the RNA solution as a template in a reverse transcriptionreaction. In producing the test cDNAs, reverse transcriptase(Superscript II; GibcoBRL) was used with 1 μl of the RNA solution,oligo-dT oligonucleotides (Amerscham Pharmacia) and the buffer providedwith the reverse transcriptase. The reverse transcription reaction wasallowed to react for 1 hour at 37° C., to provide the above the testcDNAs.

Similarly to the above 6.1.1., 1/50 by volume of the test cDNAs wereused to produce pRc/RSV-hERαG390D/S578P Kozak. In this regard, the testcDNAs were used to specifically PCR amplify therefrom witholigonucleotides depicted in SEQ ID:11 and SEQ ID:12, the cDNA encodingthe human mutant ERαG390D/S578P. The cDNA encoding the human mutantERαG390D/S578P was then PCR amplified with the oligonucleotides depictedin SEQ ID:151 and SEQ ID:12 to add a Kozak consensus sequenceimmediately upstream from the start codon (ATG) in the cDNA. Theamplified product was then inserted into the HindIII site of the plasmidpRc/RSV to provide pRc/RSV-hERαG390D/S578P Kozak.

6.2. Example 2 Production of a Plasmid Containing the Reporter Gene

An oligonucleotide depicted in SEQ ID:161 and an oligonucleotide havinga nucleotide sequence complementary thereto were synthesized with a DNAsynthesizer. The oligonucleotide depicted in SEQ ID: 161 was synthesizedto encode one of the strands of an ERE derived from the upstream regionin a Xenopus vitellogenin gene. The second oligonucleotide wassynthesized to have a nucleotide sequence complementary to theoligonucleotide depicted in SEQ ID:161. The two oligonucleotides wereannealed together to produce a DNA encoding an ERE (hereinafter referredto as the ERE DNA). The ERE DNA was then ligated together with a T4 DNAligase to provide a EREx5 DNA having a 5 tandem repeat of the ERE. A T4polynucleotide kinase was allowed to react with the EREx5 DNA tophosphorylate the ends thereof.

An oligonucleotide depicted in SEQ ID: 162 and an oligonucleotidedepicted in SEQ ID: 163 were then synthesized with a DNA synthesizer.The oligonucleotide depicted in SEQ ID: 162 was synthesized to encodeone of the strands in the nucleotide sequence of a TATA sequence derivedfrom the mouse metallothionein I gene and the leader sequence thereof.The oligonucleotide depicted in SEQ ID: 163 was synthesized to encode anucleotide sequence complementary to the oligonucleotide depicted in SEQID: 162. The oligonucleotides depicted in SEQ ID: 162 and SEQ ID:163were annealed together to produce a DNA encoding the TATA sequence. A T4polynucleotide kinase was allowed to react with the DNA encoding theTATA sequence to phosphorylate the ends thereof.

The plasmid pGL3 (Promega), which encodes the firefly luciferase gene,was restriction digested with restriction enzymes Bgl II and Hind IIIand was then treated with BAP at 65° C. for 1 hour. The restrictiondigestion reaction mixture was then subjected to low melting pointagarose gel electrophoresis (Agarose L, Nippon Gene) to confirm thatthere was a DNA fragment having the nucleotide sequence encoding thefirefly luciferase. The DNA fragment having the nucleotide sequenceencoding the firefly luciferase was then recovered from the low meltingpoint agarose gel. Subsequently, 100 ng of the recovered DNA fragmentand 1 μg of the DNA encoding the TATA sequence were used in a ligationreaction with T4 DNA ligase to provide a plasmid pGL3-TATA.

The plasmid pGL3-TATA was restriction digested with restriction enzymeSma I and was then treated with BAP at 65° C. for 1 hour. Therestriction digestion reaction mixture was then subjected to low meltingpoint agarose gel electrophoresis (Agarose L, Nippon Gene) to confirmthat there was a DNA fragment encoding the TATA sequence and the fireflyluciferase. After recovering such a DNA fragment from the low meltingpoint agarose gel, 100 ng of the recovered DNA fragment and 1 μg of theEREx5 DNA were used in a ligation reaction with T4 DNA ligase to providea plasmid pGL3-TATA-EREx5.

The plasmid pUCSV-BSD (Funakoshi) was restriction digested withrestriction enzyme BamH I to prepare a DNA encoding a blasticidin Sdeaminase gene expression cassette. Further, the plasmid pGL3-TATA-EREx5was restriction digested with restriction enzyme BamH I and was thentreated with BAP at 65° C. for 1 hour. The DNA fragment encoding ablasticidin S deaminase gene expression cassette was then mixed with therestriction digested pGL3-TATA-EREx5. The mixture was then used in aligation reaction with T4 DNA ligase to provide plasmids. The ligationreaction mixture was used to transform E. coli competent DH5α cells. Thetransformed cells are cultured in LB-amp. The clones thereof showing anampicillin resistance are then recovered. Some of the clones are thenused to isolate therefrom the plasmids derived from the ligationreaction. An aliquot sample of each of the isolated plasmids are thenrestriction digested with restriction enzyme BamH I. The restrictiondigestion reaction mixtures were then subjected to agarose gelelectrophoresis to confirm whether there was a plasmid which has astructure in which the DNA encoding a blasticidin S deaminase geneexpression cassette has been inserted into the Bam HI restriction sitein pGL3-TATA-EREx5. The plasmid having such a structure was selected andwas designated as pGL3-TATA-EREx5-BSD.

6.3. Example 3 Production of a Stably Transformed Cassette Cell

In order to produce stably transformed cassette cells, which stablycontain in one of its chromosomes the reporter gene produced in 6.2.(hereinafter referred to as the ERE reporter gene), the plasmidpGL3-TATA-EREx5-BSD was linearized and introduced into HeLa cells.

The plasmid pGL3-TATA-EREx5-BSD was restriction digested withrestriction enzyme Sal I to linearize pGL3-TATA-EREx5-BSD.

Approximately 5×10⁵ HeLa cells were cultured as host cells for 1 dayusing culture dishes having a diameter of about 10 cm (Falcon) in DMEMmedium (Nissui Pharmaceutical Co.) containing 10% FBS at 37° C. underthe presence of 5% CO₂.

The linearized pGL3-TATA-EREx5-BSD were then introduced to the culturedHeLa cells by a lipofection method using lipofectamine (LifeTechnologies). According with the manual provided with thelipofectamine, the conditions under the lipofection method included 5hours of treatment, 7 μg/dish of the plasmids above and 21 μl/dish oflipofectamine.

After the lipofection treatment, the DMEM medium was exchanged with DMEMmedium containing 10% FBS and the transformed HeLa cells were culturedfor about 36 hours. Next, the transformed HeLa cells were removed andcollected from the dish by trypsin treatment and were transferred into acontainer containing a medium to which blasticidin S was added to aconcentration of 16 μg/ml. The transformed HeLa cells were cultured insuch medium containing blasticidin S for 1 month while exchanging themedium containing blasticidin S every 3 or 4 days to a fresh batch ofthe medium containing blasticidin S.

The resulting clones, which were able to proliferate and produce acolony having a diameter of from 1 to several mm, were transferred as awhole to the wells of a 96-well ViewPlate (Berthold) to which medium hadpreviously been dispensed thereto. The colonies of the clones werefurther cultured. When the colonies proliferated to such a degree thatthey covered 50% or more of the bottom surface of the well (about 5 daysafter the transfer), the clones were removed and collected by trypsintreatment. The clones then were divided into 2 subcultures. One of thesubcultures was transferred to a 96-well ViewPlate, which was designatedas the master plate. The other subculture was transferred to a 96-wellViewPlate, which was designated as the assay plate. The master plate andthe assay plate contained medium so that the clones could be cultured.The master plate was continuously cultured under similar conditions.

After culturing the subcultures in the assay plate for 2 days, themedium was then removed from the wells of the assay plate and the clonesattached to the well walls were washed twice with PBS(−). A 5-folddiluted lysis buffer PGC50 (Toyo Ink) was added to the subcultures inthe wells of the assay plate at 20 μl per well. The assay plate was leftstanding at room temperature for 30 minutes and were set on aluminometer LB96P (Berthold), which was equipped with an automaticsubstrate injector. Subsequently, 50 μl of the substrate solution PGL100(Toyo Ink) was automatically dispensed to each of the lysed clones inthe assay plate to measure the luciferase activity therein with theluminometer LB96P. Ten (10) clones, which exhibited a high luciferaseactivity were selected therefrom.

Samples of the clones in the master plate, which correspond to theselected 10 clones were then cultured at 37° C. for 1 to 2 weeks in thepresence of 5% CO₂ using dishes having a diameter of about 10 cm(Falcon) in medium.

The plasmid pRc/RSV-hERαKozak was then introduced to the selected clonesby a lipofection method using lipofectamine (Life Technologies) toprovide a second round of clones. According with the manual providedwith the lipofectamine, the conditions under the lipofection methodincluded 5 hours of treatment, 7 μg/dish of the plasmids above and 21μl/dish of lipofectamine. A DMSO solution containing 17β-E2 was thenadded to the resulting second clones to a concentration of 10 nM. Afterculturing the second clones for 2 days, the luciferase activity wasmeasured, similarly to the above, for each of the second clones. Theclone in the master plate, which provided the second clone exhibitingthe highest induction of luciferase activity, was selected as the stablytransformed cassette cell which stably contained in one of itschromosomes the ERE reporter gene (hereinafter referred to as the stablytransformed ERE cassette cell).

6.4. Example 4 Production of Stably Transformed Binary Cells

Four stably transformed cells containing the ERE reporter gene with thehuman mutant ERαG390D, S578P or G390D/S578P or human normal ERα(hereinafter referred to as the stably transformed ERE binary cells)were produced. The first stably transformed ERE binary cell contained inits chromosomes the linearized pGL3-TATA-EREx5-BSD, which encodes thereporter gene, and the linearized pRc/RSV-hERαKozak, which encodes thehuman normal ERα. The second stably transformed ERE binary cellcontained in its chromosomes the linearized pGL3-TATA-EREx5-BSD, whichencodes the ERE reporter gene, and the linearized pRc/RSV-hERαG390DKozak, which encodes the human mutant ERαG390D. The third stablytransformed ERE binary cell contained in its chromosomes the linearizedpGL3-TATA-EREx5-BSD, which encodes the ERE reporter gene, and thelinearized pRc/RSV-hERαS578P Kozak, which encodes the human mutantERαS578P. The fourth stably transformed ERE binary cell contained in itschromosomes the linearized pGL3-TATA-EREx5-BSD, which encodes the EREreporter gene, and the linearized pRc/RSV-hERαG390D/S578P Kozak, whichencodes the human mutant ERαG390D/S578P.

In order to produce the stably transformed ERE binary cells, theplasmids pGL3-TATA-EREx5-BSD, pRc/RSV-hERαG390D Kozak, pRc/RSV-hERαS578PKozak and pRc/RSV-hERαG390D/S578P Kozak were each linearized andintroduced into HeLa cells. To linearize, the plasmids above wererestriction digested with restriction enzyme Sal I.

Approximately 5×10⁵ HeLa cells were cultured as host cells for 1 dayusing dishes having a diameter of about 10 cm (Falcon) in DMEM medium(Nissui Pharmaceutical Co.) containing 10% FBS at 37° C. under thepresence of 5% CO₂.

A linearized pGL3-TATA-EREx5-BSD was introduced, respectively, into theHeLa cells with a linearized plasmid encoding a human mutant ERα orhuman normal ERα, as shown in Table 6 below. The linearized plasmidswere introduced into the HeLa cells by a lipofection method usinglipofectamine (Life Technologies). According with the manual providedwith the lipofectamine, the conditions for each of the treatments underthe lipofection method included 5 hours of treatment, 7 μg/dish of theplasmids (3.5 μg each) and 21 μl/dish of lipofectamine.

TABLE 6 linearized plasmids first HeLa cells pGL3-TATA-EREx5-BSD andpRc/RSV-hERαKozak second HeLa cells pGL3-TATA-EREx5-BSD andpRc/RSV-hERαG390D Kozak third HeLa cells pGL3-TATA-EREx5-BSD andpRc/RSV-hERαS578P Kozak fourth HeLa cells pGL3-TATA-EREx5-BSD andpRc/RSV-hERαG390D/S578P Kozak

After the lipofection treatment, the DMEM mediums were exchanged withDMEM medium containing 10% FBS and the transformed HeLa cells werecultured for about 36 hours. Next, the transformed HeLa cells wereremoved and collected, respectively, from the dishes by trypsintreatment and were transferred into a container containing a medium towhich blasticidin S and G418 was added thereto. The concentration of theblasticidin S therein for each of the cell cultures was 16 μg/ml. Theconcentration of the G418 therein for each of the cell cultures was 800μg/ml. The transformed HeLa cells were cultured in such mediumcontaining blasticidin S and G418 for 1 month while exchanging themedium every 3 or 4 days to a fresh batch of the medium containingblasticidin S and G418.

The resulting clones, which were able to proliferate to a diameter offrom 1 to several mm, were transferred, respectively, to the wells of96-well ViewPlates (Berthold) to which medium had previously beendispensed thereto. The clones were further cultured. When the clonesproliferated to such a degree that they covered 50% or more of thebottom surface of the well (about 5 days after the transfer), the cloneswere removed and collected by trypsin treatment. Each of the clones thenwere divided into 3 subcultures. For each of the clones, one of thesubcultures was transferred to a 96-well ViewPlate, which was designatedas the master plate. The other two subcultures were transferred,respectively, to 96-well ViewPlates, which were designated as the assayplates. The master plate and the assay plates contained medium so thatthe clones can be cultured. The master plate is continuously culturedunder similar conditions. To each of the subcultures in the first assayplate, a DMSO solution containing 17β-E2 was added to a concentration of10 nM. An equivalent volume of DMSO was added to the subcultures in thesecond assay plate. The first and second assay plates were then culturedfor 2 days.

The medium was then removed from the wells of the first and second assayplates and the clones attached to the well walls were washed twice withPBS(−). A 5-fold diluted lysis buffer PGC50 (Toyo Ink) was added to theclones in the wells of the first and second assay plates at 20 μl perwell. The first and second assay plates were left standing at roomtemperature for 30 minutes and were set on a luminometer LB96P(Berthold), which was equipped with an automatic substrate injector.Subsequently, 50 μl of the substrate solution PGL100 (Toyo Ink) wasautomatically dispensed, respectively, to each of the lysed clones inthe assay plates to measure the luciferase activity therein with theluminometer LB96P. Clones in master plate corresponding to the clones inthe first assay plate which exhibited a 2-fold higher induction ofluciferase activity (%) were then selected as the stably transformed EREbinary cell which stably contain the reporter gene with the human mutantERαG390D, S578P or G390D/S578P gene or the human normal ERα gene.

6.5. Example 5 Reporter Assays of Human Mutant ERα 6.5.1 Preparation ofthe Stably Transformed ERE Binary Cells for the Reporter Assay

About 2×10⁴ cells of the stably transformed ERE binary cells, producedin the above 6.4., were then transferred to the wells of 96-wellLuminometer plates (Corning Coaster) to culture overnight the stablytransformed ERE binary cells in an E-MEM medium to which a charcoaldextran treated FBS was added to a concentration of 10% (v/v)(hereinafter referred to as charcoal dextran FBS/E-MEM medium).

6.5.2. Introduction of the Plasmids Encoding the Human Mutant ERαK303R,S309F, M396V, G415V, G494V or K531E

Seven subcultures which contained, respectively, approximately 2×10⁶cells of the stably transformed ERE cassette cells produced in 6.3.,were cultured for 1 day using dishes having a diameter of about 10 cm(Falcon) in charcoal dextran FBS/E-MEM medium.

For transient expression, the plasmid pRc/RSV-hERαKozak (produced in6.1.1., encoding normal ERα) and a plasmid encoding the mutant ERα(produced in 6.1.2.2., i.e., pRc/RSV-hERαK303R Kozak, pRc/RSV-hERαS309FKozak, pRc/RSV-hERαM396V Kozak, pRc/RSV-hERαG415V Kozak,pRc/RSV-hERαG494V Kozak or pRc/RSV-hERαK531E Kozak, each encoding ahuman mutant ERα) were introduced, respectively, into the subcultures ofthe stably transformed ERE cassette cells by a lipofection method usinglipofectamine (Life Technologies). According with the manual providedwith the lipofectamine, the conditions for each of the treatments underthe lipofection method included 5 hours of treatment, 7 μg/dish of theplasmids and 21 μl/dish of lipofectamine. After culturing the resultingcell subcultures at 37° C. for 16 hours in the presence of 5% CO₂, thecharcoal dextran FBS/E-MEM medium therein was exchanged to fresh batchesof the charcoal dextran FBS/E-MEM medium to further culture each of thecell subcultures for 3 hours. The cell subcultures were then collected,respectively, and uniformly suspended in charcoal dextran FBS/E-MEMmedium.

6.5.3. Measurement of the Activity for Transactivation of the ReporterGene

Four (4) general types of DMSO solutions were used to expose the cellsin the subcultures prepared in the above 6.5.1. and 6.5.2. with variousconcentrations of a pure or partial anti-estrogen. The first DMSOsolutions were prepared to contain a partial anti-estrogen(4-hydroxytamoxifen or raloxifene) at various concentrations. The secondDMSO solutions were prepared to contain a pure anti-estrogen (ZM189154)at various concentrations. The third DMSO solutions were prepared tocontain E2 at 10 nM and a partial anti-estrogen (4-hydroxytamoxifen orraloxifene) at various concentrations. The fourth DMSO solutions wereprepared to contain E2 at 10 nM and a pure anti-estrogen (ZM189154) atvarious concentrations.

The first, second, third or fourth DMSO solution was then added to thesubcultures prepared, in the above 6.5.1. and 6.5.2., as shown in Tables7, 8, 9 and 10 below. The first, second, third or fourth DMSO solutionwas added to the wells of the 96-well ViewPlates such that theconcentration of the first, second, third or fourth DMSO solution ineach of the wells was about 0.1% (v/v). Further, 2 controls wereprepared for each of the subcultures in the wells of a 96-wellViewPlate. One of the controls was exposed to DMSO (containing nopartial or pure anti-estrogen). The second control was exposed to a DMSOsolution consisting essentially of 100 pM of E2.

The cells were then cultured for 36 to 40 hours at 37° C. in thepresence of 5% CO₂. A 5-fold diluted lysis buffer PGC50 (Toyo Ink) wasadded, respectively, to the cells in the wells at 50 μl per well. The96-well ViewPlates were periodically and gently shook while beingincubated at room temperature for 30 minutes. Ten microliters (10 μl) ofthe lysed cells were then transferred, respectively, to white 96-wellsample plates (Berthold) and were set on a luminometer LB96P (Berthold),which was equipped with an automatic substrate injector. Subsequently,50 μl of the substrate solution PGL100 (Toyo Ink) was automaticallydispensed, respectively, to each of the lysed cells in the white 96-wellsample plates to instantaneously measure for 5 seconds the luciferaseactivity therein with the luminometer LB96P.

The luciferase activities resulting from the cells prepared in 6.5.2.are illustrated in FIGS. 1 to 32.

FIGS. 1 and 2 illustrate the luciferase activity provided by the humannormal ERα and the human mutant ERαK303R in the presence of4-hydroxytamoxifen or ZM189154 as the sole possible agent of stimulatingthe human normal ERα or the human mutant ERαK303R.

FIG. 3 illustrates the luciferase activity provided by the human normalERα and the human mutant ERαK303R in the presence of E2 with ZM189154.

FIGS. 4 and 5 illustrate the luciferase activity provided by the humannormal ERα and the human mutant ERαS309F in the presence of4-hydroxytamoxifen or ZM189154 as the sole possible agent of stimulatingthe human normal ERα or the human mutant ERαS309F.

FIG. 6 illustrates the luciferase activity provided by the human normalERα and the human mutant ERαS309F in the presence of E2 with ZM189154.

FIGS. 7 and 8 illustrate the luciferase activities provided by the humannormal ERα and the human mutant ERαM396V in the presence of4-hydroxytamoxifen or raloxifene as the sole possible agent ofstimulating the human normal ERα or the human mutant ERαM396V.

FIGS. 9 to 11 illustrate the luciferase activity provided by the humannormal ERα and the human mutant ERαM396V in the presence of E2 with4-hydroxytamoxifen, raloxifene or ZM189154.

FIGS. 12 and 13 illustrate the luciferase activity provided by the humannormal ERα and the human mutant ERαG415V in the presence of4-hydroxytamoxifen or ZM189154 as the sole possible agent of stimulatingthe human normal ERα or the human mutant ERαG415V.

FIGS. 14 and 15 illustrate the luciferase activity provided by the humannormal ERα and the human mutant ERαG415V in the presence of E2 with4-hydroxytamoxifen or ZM189154.

FIGS. 16 to 17 illustrate the luciferase activity provided by the humannormal ERα and the human mutant ERαG494V in the presence of4-hydroxytamoxifen or raloxifene as the sole possible agent ofstimulating the human normal ERα or the human mutant ERαG494V.

FIGS. 18 to 20 illustrate the luciferase activity provided by the humannormal ERα and the human mutant ERαG494V in the presence of E2 with4-hydroxytamoxifen, raloxifene or ZM189154.

FIGS. 21 to 26 illustrate the luciferase activity provided by the humannormal ERα and the human mutant ERαK531E in the presence of4-hydroxytamoxifen, raloxifene or ZM189154 as the sole possible agent ofstimulating the human normal ERα or the human mutant ERαK531E.

FIGS. 27 to 32 illustrate the luciferase activity provided by the humannormal ERα and the human mutant ERαK531E in the presence of E2 with4-hydroxytamoxifen, raloxifene or ZM189154.

The luciferase activities resulting from the cells prepared in 6.5.1.are illustrated in FIGS. 33 to 48.

FIGS. 33 to 40 illustrate the luciferase activity provided by the humannormal ERα, human mutant ERαG390D, human mutant ERαS578P and humanmutant ERαG390D/S578P in the presence of 4-hydroxytamoxifen, raloxifeneor ZM189154 as the sole probable agent of stimulating the human normalERα, human mutant ERαG390D, human mutant ERαS578P and human mutantERαG390D/S578P.

FIGS. 41 to 48 illustrate the luciferase activity provided by the humannormal ERα, human mutant ERαG390D, human mutant ERαS578P and humanmutant ERαG390D/S578P in the presence of E2 with 4-hydroxytamoxifen,raloxifene or ZM189154.

TABLE 7 utilized plasmid for human normal DMSO exposed partial or ormutant ERα solution pure anti-estrogen 1 pRC/RSV-hERαKozak first4-hydroxytamoxifen 2 pRC/RSV-hERaαK303R Kozak first 4-hydroxytamoxifen 3pRC/RSV-hERαKozak second ZM189154 4 pRC/RSV-hERαK303R Kozak secondZM189154 5 pRC/RSV-hERαKozak fourth ZM189154 6 pRC/RSV-hERαK303R Kozakfourth ZM189154 7 pRC/RSV-hERαKozak first 4-hydroxytamoxifen 8pRC/RSV-hERαS309F Kozak first 4-hydroxytamoxifen 9 pRC/RSV-hERαKozaksecond ZM189154 10 pRC/RSV-hERαS309F Kozak second ZM189154 11pRC/RSV-hERαKozak fourth ZM189154 12 pRC/RSV-hERαS309F Kozak fourthZM189154

TABLE 8 utilized plasmid for human normal DMSO exposed partial or ormutant ERα solution pure anti-estrogen 13 pRC/RSV-hERαKozak first4-hydroxytamoxifen 14 pRC/RSV-hERαM396V Kozak first 4-hydroxytamoxifen15 pRC/RSV-hERαKozak first raloxifene 16 pRC/RSV-hERαM396V Kozak firstraloxifene 17 pRC/RSV-hERαKozak third 4-hydroxytamoxifen 18pRC/RSV-hERαM396V Kozak third 4-hydroxytamoxifen 19 pRC/RSV-hERαKozakthird raloxifene 20 pRC/RSV-hERαM396V Kozak third raloxifene 21pRC/RSV-hERαKozak fourth ZM189154 22 pRC/RSV-hERαM396V Kozak fourthZM189154 23 pRC/RSV-hERαKozak first 4-hydroxytamoxifen 24pRC/RSV-hERαG415V Kozak first 4-hydroxytamoxifen 25 pRC/RSV-hERαKozaksecond ZM189154 26 pRC/RSV-hERαG415V Kozak second ZM189154 27pRC/RSV-hERαKozak third 4-hydroxytamoxifen 28 pRC/RSV-hERαG415V Kozakthird 4-hydroxytamoxifen 29 pRC/RSV-hERαKozak fourth ZM189154 30pRC/RSV-hERαG415V Kozak fourth ZM189154

TABLE 9 utilized plasmid for human normal DMSO exposed partial or ormutant ERα solution pure anti-estrogen 31 pRC/RSV-hERαKozak first4-hydroxytamoxifen 32 pRC/RSV-hERαG494V Kozak first 4-hydroxytamoxifen33 pRC/RSV-hERαKozak first raloxifene 34 pRC/RSV-hERαG494V Kozak firstraloxifene 35 pRC/RSV-hERαKozak third 4-hydroxytamoxifen 36pRC/RSV-hERαG494V Kozak third 4-hydroxytamoxifen 37 pRC/RSV-hERαKozakthird raloxifene 38 pRC/RSV-hERαG494V Kozak third raloxifene 39pRC/RSV-hERαKozak fourth ZM189154 40 pRC/RSV-hERαG494V Kozak fourthZM189154 41 pRC/RSV-hERαKozak first 4-hydroxytamoxifen 42pRC/RSV-hERαK531E Kozak first 4-hydroxytamoxifen 43 pRC/RSV-hERαKozakfirst raloxifene 44 pRC/RSV-hERαK531E Kozak first raloxifene 45pRC/RSV-hERαKozak second ZM189154 46 pRC/RSV-hERαK531E Kozak secondZM189154 47 pRC/RSV-hERαKozak third 4-hydroxytamoxifen 48pRC/RSV-hERαK531E Kozak third 4-hydroxytamoxifen 49 pRC/RSV-hERαKozakthird raloxifene 50 pRC/RSV-hERαK531E Kozak third raloxifene 51pRC/RSV-hERαKozak fourth ZM189154 52 pRC/RSV-hERαK531E Kozak fourthZM189154

TABLE 10 human normal or mutant ERα DMSO exposed partial or encoded inthe chromosomes solution pure anti-estrogen 53 human normal ERα first4-hydroxytamoxifen 54 human mutant ERαG390D first 4-hydroxytamoxifen 55human mutant ERαS578P first 4-hydroxytamoxifen 56 human mutantERαG390D/S578P first 4-hydroxytamoxifen 57 human normal ERα firstraloxifene 58 human mutant ERαG390D/S578P first raloxifene 59 humannormal ERα second ZM189154 60 human mutant ERαG390D/S578P secondZM189154 61 human normal ERα third 4-hydroxytamoxifen 62 human mutantERα390D third 4-hydroxytamoxifen 63 human mutant ERαS578P third4-hydroxytamoxifen 64 human mutant ERαG390D/S578P third4-hydroxytamoxifen 65 human normal ERα third raloxifene 66 human mutantERαG390D/S578P third raloxifene 67 human normal ERα fourth ZM189154 68human mutant ERαG390D/S578P fourth ZM189154

6.6. Example 6 Comparative Dually Transient Reporter Assay

Approximately 2×10⁶ HeLa cells were cultured for 1 day using disheshaving a diameter of about 10 cm (Falcon) in charcoal dextran FBS/E-MEMmedium at 37° C. in the presence of 5% CO₂. After culturing the HeLacells, the HeLa cells were divided into two subcultures.

Subsequently, 3.75 μg of pRc/RSV-hERαKozak and 3.75 μg ofpGL3-TATA-EREx5 were introduced into the HeLa cells in the firstsubculture by a lipofection method using lipofectamine for transientexpression. In the second subculture, 3.75 μg of pRc/RSV-hERαK531E Kozakand 3.75 μg of pGL3-TATA-EREx5 were introduced by the lipofection methodusing lipofectamine for transient expression. The first and secondsubcultures were then cultured at 37° C. for 16 hours in the presence of5% CO₂. After exchanging the charcoal dextran FBS/E-MEM medium thereinwith a fresh batch of charcoal dextran FBS/E-MEM medium the first andsecond subcultures were then similarly cultured for 3 hours. The cellsin the first and second subcultures were then collected, respectively,and were uniformly suspended in charcoal dextran FBS/E-MEM medium.

Two (2) general types of DMSO solutions were prepared to expose thecells in the first and second subcultures. The first DMSO solutions wereprepared to contain 4-hydroxytamoxifen at various concentrations. Thesecond DMSO solutions were prepared to contain 10 nM of E2 and4-hydroxytamoxifen at various concentrations.

The first and second DMSO solutions were then nixed, respectively, withthe first and second subcultures in 96-well ViewPlates such that theconcentration of the first or second DMSO solution in each of the wellswas about 0.1% (v/v).

The first and second subcultures were then cultured for 36 hours at 37°C. in the presence of 5% CO₂. A 5-fold diluted lysis buffer PGC50(Nippon Gene) was added, respectively, to the first and secondsubcultures in the wells at 50 μl per well. The 96-well ViewPlates wereperiodically and gently shook while being incubated at room temperaturefor 30 minutes. Ten microliters (10 μl) of the resulting lysed cellswere then transferred, respectively, to white 96-well sample plates(Berthold) and were set on a luminometer LB96P (Berthold), which wasequipped with an automatic substrate injector. Subsequently, 50 μl ofthe substrate solution PGL100 (Toyo Ink) was automatically dispensed,respectively, to each of the lysed cells in the white 96-well sampleplates to instantaneously measure for 5 seconds the luciferase activitytherein with the luminometer LB96P.

The luciferase activity from the dually transient reporter assay areshown in FIGS. 49 to 52.

FIGS. 49 and 50 illustrate the luciferase activity provided by the humannormal ERα and human mutant ERαK531E in the presence of4-hydroxytamoxifen as the sole probable agent of stimulating the humannormal ERα or human mutant ERαK531E.

FIGS. 51 and 52 illustrate, respectively, the luciferase activity ofmutant human normal ERα and human mutant ERαK531E in the presence of E2with 4-hydroxytamoxifen.

6.7. Example 7 Search Oligonucleotides

In order to search for a variant codon that encodes a substituted aminoacid in a human test ERα, search oligonucleotides are designed so thatthe search oligonucleotides can anneal to a searching region in a humantest ERα gene when the human test ERα gene encodes a human normal ERα.The searching regions include the codon encoding the amino acid atrelative position 303, the codon encoding the amino acid at relativeposition 309, the codon encoding the amino acid at relative position390, the codon encoding the amino acid at relative position 396, thecodon encoding the amino acid at relative position 415, the codonencoding the amino acid at relative position 494, the codon encoding theamino acid at relative position 531 or the codon encoding the amino acidat relative position 578. Further the oligonucleotides are designed tohave a GC content of from 30% to 70% and a size of 20 bp. Based on theoligonucleotides so designed, the oligonucleotides of the presentinvention indicated by the above are synthesized with a DNA synthesizer(Model 394, Applied Biosystems).

6.8 Example 8 Genotype Diagnosis by PCR Amplification and NucleotideSequencing Methods

A test human liver tissue sample is used to diagnose the genotype of thetest ERα polynucleotide therein. In utilizing the test human livertissue sample, 0.1 g of the test human liver tissue sample ishomogenized with a homogenizer in 5 ml of a buffer containing 4Mguanidium thiocyanate, 0.1M Tris-HCl (pH 7.5) and 1% β mercaptoethanol.The resulting buffer is layered with 25 ml of an aqueous 5.7M CsClsolution and is ultracentrifuged at 90,000×g for 24 hours to obtain aRNA pellet. After rinsing the RNA pellet with 70% ethanol, the RNApellet is allowed to air dry at room temperature. The RNA pellet is thendissolved in 10 μl of sterile water to a concentration of 1.2 μg/ml. Asolution of test cDNAs is then produced by collectively using the RNAsin the RNA solution as a template in a reverse transcription reaction.In producing the test cDNAs, Superscript II (Gibco) was used with 1 μlof the RNA solution, oligo-dT oligonucleotides (Amerscham-Pharmacia) andthe buffer provided with the oligo-dT oligonucleotides. The reversetranscription reaction was allowed to react for 1 hour at 37° C.

Using 1/50 by volume samples of the test cDNAs, a PCR amplification isconducted with combinations of the search oligonucleotides shown inTable 11 below.

TABLE 11 Search Oligonucleotides 1 SEQ ID: 32 and SEQ ID: 38 2 SEQ ID:42 and SEQ ID: 48 3 SEQ ID: 52 and SEQ ID: 58 4 SEQ ID: 62 and SEQ ID:68 5 SEQ ID: 72 and SEQ ID: 78 6 SEQ ID: 82 and SEQ ID: 88 7 SEQ ID: 92and SEQ ID: 98 8 SEQ ID: 109 and SEQ ID: 110The PCR mixtures in these PCR amplifications contain the test cDNAs,AmpliTaq DNA polymerase (Perkin Elmer), 100 μM of dNTPs (dATP, dTTP,dGTP, dCTP), one of the combinations of the search oligonucleotides andthe buffer provided with the AmpliTaq Polymerase. In this PCRamplification, there are repeated 35 times for each of the PCRamplifications, an incubation cycle entailing an incubation at 95° C.for 1 minute, then an incubation at 55° C. for 30 sec, which is followedby an incubation at 72° C. for 1 minute. The obtained searching regionpolynucleotides are subjected to 1% low melting point agarose gelelectrophoresis (Agarose L, Nippon Gene) and are recovered. Using wholeamounts of the recovered searching region polynucleotides, the searchingregions are sequenced. The nucleotide sequences of the searching regionsare compared to the nucleotide sequence encoding human normal ERα.

6.9. Example 9 Genotype Diagnosis by SSCP Methods 6.9.1. Extraction ofTest Genomic DNAs from a Test Tissue Sample

Test genomic DNAs from a test tissue sample is prepared by the methodsdescribed in TAKARA PCR Technical news No. 2, Takara Shuzo (September1991). This procedure in relation with the present invention isdescribed below.

Two (2) to 3 hair samples from a test subject are washed with sterilewater and then 100% ethanol. After air drying the hair samples, the hairsamples are cut to 2 to 3 mm and are transferred to a plastic test tube.Two hundred microliters (200 μl) of BCL buffer (10 mM Tris-HCl (pH.7.5), 5 mM MgCl₂, 0.32M sucrose, 1% Triton X-100) are added thereto.Subsequently, a Proteinase K solution and a SDS solution are mixedtherewith to amount to 100 μg/ml and 0.5% (w/v), respectively.

After incubating the resulting mixture for 1 hour at 70° C., the mixtureis phenol-chloroform extracted to recover the aqueous layer therefrom.In the phenol-chloroform extraction, a substantially equal volume ofphenol-chloroform is added to the mixture. The mixture is shakenvigorously and is centrifuged (15,000 rpm, 20,000×g, 5 min, 4° C.). Theaqueous layer therefrom is extracted with a pipette so that the phenollayer is not disturbed. A second phenol-chloroform extraction is thensimilarly conducted with the aqueous layer.

A substantially equal volume of chloroform is mixed with the aqueouslayer from the second phenol-chloroform extraction, to extract theaqueous layer from the resulting chloroform mixture. In this extractionwith chloroform, the chloroform mixture is shaken vigorously and iscentrifuged, so that the aqueous layer can be extracted from thechloroform mixture. Five hundred microliters (500 μl) of 100% ethanol isthen added to the aqueous layer from the chloroform mixture. The testgenomic DNAs therein is precipitated at −80° C. for 20 minutes and isthen centrifuged to obtain a pellet of the test genomic DNAs. Theobtained pellet of the test genomic DNAs is dried and dissolved insterile water, to so that test genomic DNAs can provide a test ERαpolynucleotide.

Alternatively, peripheral blood can be used as a test sample from whichtest genomic DNAs can be obtained. Ten milliliters (10 ml) of blood iscollected from a test subject and test genomic DNAs are extracted fromthe blood, using a DNA Extraction kit (Stratagene).

6.9.2. Analysis of Test Genomic DNA by the PCR-SSCP Method

Combinations of a forward search oligonucleotide and a reverse searcholigonucleotide are selected for PCR amplifications with the testgenomic DNAs. The combinations of the forward and reverse searcholigonucleotide are selected, based on the locus of the searchingregions in the test ERα polynucleotide. The combinations of the forwardand reverse search oligonucleotides in connection with the searchingregions which are suspected to contain the valiant codon encoding asubstituted amino acid at the provided relative positions are shown inTable 12 below.

TABLE 12 searching Forward search Reverse search region oligonucleotideoligonucleotide relative SEQ ID: 29, SEQ ID: 30, SEQ ID: 34, SEQ ID: 35,position 303 SEQ ID: 31, SEQ ID: 32 or SEQ ID: 36, SEQ ID: 37 or SEQ ID:33 SEQ ID: 38 relative SEQ ID: 39, SEQ ID: 40, SEQ ID: 44, SEQ ID: 45,position 309 SEQ ID: 41, SEQ ID: 42 or SEQ ID: 46, SEQ ID: 47 or SEQ ID:43 SEQ ID: 48 relative SEQ ID: 49, SEQ ID: 50, SEQ ID: 54, SEQ ID: 55,position 390 SEQ ID: 51, SEQ ID: 52 or SEQ ID: 56, SEQ ID: 57 or SEQ ID:53 SEQ ID: 58 relative SEQ ID: 59, SEQ ID: 60, SEQ ID: 64, SEQ ID: 65,position 396 SEQ ID: 61, SEQ ID: 62 or SEQ ID: 66, SEQ ID: 67 or SEQ ID:63 SEQ ID: 68 relative SEQ ID: 69, SEQ ID: 70, SEQ ID: 74, SEQ ID: 75,position 415 SEQ ID: 71, SEQ ID: 72 or SEQ ID: 76, SEQ ID: 77 or SEQ ID:73 SEQ ID: 78 relative SEQ ID: 79, SEQ ID: 80, SEQ ID: 84, SEQ ID: 85,position 494 SEQ ID: 81, SEQ ID: 82 or SEQ ID: 86, SEQ ID: 87 or SEQ ID:83 SEQ ID: 88 relative SEQ ID: 89, SEQ ID: 90, SEQ ID: 94, SEQ ID: 95,position 531 SEQ ID: 91, SEQ ID: 92 or SEQ ID: 96, SEQ ID: 97 or SEQ ID:93 SEQ ID: 98 relative SEQ ID: 99, SEQ ID: 100, SEQ ID: 104, SEQposition 578 SEQ ID: 101, SEQ ID: 102 ID: 105, SEQ ID: 106, or SEQ ID:103 SEQ ID: 107 or SEQ ID: 108

The combinations of the forward and reverse search oligonucleotides aresynthesized with a DNA synthesizer. Each of the forward and reversesearch oligonucleotides are labeled with ³²P using a DNA MEGALABEL kit(Takara Shuzo). The test genomic DNAs are then used, respectively, inthe PCR amplifications to provide amplified searching regionpolynucleotides. Each of the PCR mixtures in these PCR amplificationscontain Amplitaq DNA Polymerase (Perkin Elmer), 400 μM of dNTPs (100 μMof dATP, 100 μM of dTTP, 100 μM of dGTP and 100 μM of dCTP), 100 pmol ofthe ³²P labeled forward search oligonucleotide, 100 pmol of the ³²Plabeled reverse search oligonucleotide, 1 μg of the test genomic DNA andthe buffer provided with the Amplitaq DNA Polymerase. In each of thesePCR amplifications, there are repeated 35 times for each of the PCRamplifications, an incubation cycle entailing an incubation at 94° C.for 1 minute, then an incubation at 55° C. for 30 seconds, which isfollowed by an incubation at 72° C. for 1 minute.

After the PCR amplifications, 1/20 by volume samples from each of theamplified searching region polynucleotides are heat denatured in 80%formamide at 80° C. for 5 minutes. Subsequently, each the heat denaturedsearching region polynucleotides are subjected to electrophoresis in 5%native polyacrylamide gels using 180 mM Tris-borate buffer (pH 8.0). Theconditions for electrophoresis include a room temperature air coolingand a constant power of 40 W for 60 min. After electrophoresis, the 5%native polyacrylamide gels are autoradiographed using X-ray films byusing conventional procedures to detect the radioactivity of thesearching regions.

Since a product encoding the valiant codon has a different mobility inthe 5% native polyacrylamide gel as compared with a product encoding anormal codon, a comparison of each of the mobilities of the searchingregion polynucleotides with a standard polynucleotide encoding acorresponding region in a human normal ERα detects the presence orabsence of a mutation in the searching regions.

6.9.3. Determination of Mutation

After detecting a variant codon in the searching regions, 1 mm squareportions containing the searching region polynucleotides are cut out ofthe 5% native polyacrylamide gels. Each of the 1 mm square portions aretreated at 90° C. for 10 min in 100 μl of sterile water to recover thesearching region polynucleotides from the 1 mm square portions.Subsequently, 1/20 by volume samples of the searching regionpolynucleotides are then used, respectively, in a second round of PCRamplifications. The oligonucleotides in these PCR amplifications usedthe combinations of the search oligonucleotides used in the above 6.9.2.Each of the PCR mixtures in these PCR amplifications contain AmplitaqDNA Polymerase (ABI), 400 μM of dNTPs (100 μM of dATP, 100 μM of dTTP,100 μM of dGTP and 100 μM of dCTP), the forward search oligonucleotide,the reverse search oligonucleotide, one of the test DNA fragments andthe buffer provided with the Amplitaq DNA polymerase. In each of thesePCR amplifications, there are repeated 35 times, an incubation cycleentailing an incubation at 94° C. for 1 minute, then an incubation at55° C. for 30 seconds, which is followed by an incubation at 72° C. for1 minute.

After completion of the reaction, the amplified searching regionpolynucleotides are subjected to low melting point agarose gelelectrophoresis (Agarose L: Nippon Gene). After recovering the amplifiedsearching region polynucleotides from the low melting point agarosegels, the recovered searching region polynucleotides are prepared with aDye Terminator cycle sequence ready reaction kit (Applied Biosystems).The prepared sample of the searching region polynucleotides aresequenced, respectively, with an ABI autosequencer (Model 377, AppliedBiosystems), to determine the mutation in the valiant codons, ifpresent, in the searching regions.

6.10. Example 10 Genotype Diagnosis by RFLP Methods

Combinations of a forward search oligonucleotide and a reverse searcholigonucleotide are selected for PCR amplifications with test genomicDNAs or a test cDNAs. The combinations of the forward and reverse searcholigonucleotides are selected, based on the locus of the searchingregions in the test ERα polynucleotide. The combinations of the forwardand reverse search oligonucleotides are shown in Table 13 below, inconnection with the searching regions which are suspected to contain avariant codon encoding a substituted amino acid at the provided relativepositions in Table 13 below.

TABLE 13 Searching region oligonucleotides Relative position 303 SEQ ID:164 and SEQ ID: 165 Relative position 309 SEQ ID: 166 and SEQ ID: 167Relative position 396 SEQ ID: 168 and SEQ ID: 169 Relative position 415SEQ ID: 170 and SEQ ID: 171 Relative position 494 SEQ ID: 172 and SEQID: 173 Relative position 531 SEQ ID: 174 and SEQ ID: 175The test genomic DNAs or test cDNAs are used in the PCR amplificationsto provide amplified searching region polynucleotides having a size ofabout 100 or 160 bp. Each of the PCR mixtures in these PCRamplifications contain Amplitaq DNA Polymerase (ABI), the test genomicDNAs or test cDNAs, dNTPs (dATP, dTTP, dGTP and dCTP), the forwardsearch oligonucleotide, the reverse search oligonucleotide and thebuffer provided with the Amplitaq DNA Polymerase. In each of these PCRamplifications, there are repeated 35 times, an incubation cycleentailing an incubation at 94° C. for 1 minute, then an incubation at55° C. for 30 seconds, which is followed by an incubation at 72° C. for1 minute.

Samples of each of the searching region polynucleotides are then mixed,respectively, with various restriction enzymes for restriction digestionreactions (one restriction enzyme per restriction digestion reaction)and are incubated at 37° C. at 1 hour. The restriction digestionreaction mixtures are subjected to agarose gel electrophoresis toconfirm whether the searching region polynucleotides are successfullyrestriction digested with one of the various restriction enzymes. Asuccessful restriction digest with the restriction enzymes shown inTable 14 and Table 15 below indicate whether there is in the searchingregion, a valiant codon encoding a substituted amino acid at theprovided relative position in Table 14 and Table 15 below

TABLE 14 relative relative position 309 position 494 restriction enzymeApa I Stu I approximate length of 100 bp 150 bp searching region whenencoding normal codon restriction digestion yes yes approximate lengthof 40 bp/60 bp 100 bp/50 bp resulting DNA fragments

In reference to Table 14, an unsuccessful restriction digestion with theprovided restriction enzyme at the codon encoding the amino acid at theprovided relative position, indicates that such a codon is a valiantcodon. In such cases, the searching regions are sequenced with an ABIautosequencer (Model 377, Applied Biosystems) to determine the mutationin the variant codons, if present, in the searching regions.

TABLE 15 relative relative relative relative position position positionposition 303 396 415 531 restriction enzyme: Stu I ApaL I Kpn I Sac Iapproximate length of 100 bp 100 bp 100 bp 100 bp searching region: whenencoding normal codon restriction digestion no no no no approximatelength of — — — — resulting DNA fragments: when encoding variant codonwhen variant codon AGG GTG GTA GAG sequence is: restriction digestion:yes yes yes yes approximate length of 40 bo/ 40 bo/ 40 bo/ 40 bo/resulting DNA fragments: 60 bp 60 bp 60 bp 60 bp

In reference to Table 15, a successful restriction digestion with theprovided restriction enzyme at the codon encoding the amino acid at theprovided relative position, indicates that such a codon is a valiantcodon. In such cases, it is determined that the mutations in the variantcodons, if present, are the nucleotide sequences provided in the aboveTable 15.

6.11. Example 11 Genotype Diagnosis by Southern Hybridization Methods

Five micrograms (5 μg) of test genomic DNA, provided in 6.9.1., isthoroughly restriction digested with the restriction enzyme Stu I. Therestriction digestion reaction mixture is subjected to electrophoresisat 20V for 16 hours with a 4% Nusieve 3:1 agarose gel (FMC BIO). Thecapillary alkali blotting method (Hybond blotting membrane manual,Amerscham) is used to blot for 2 hours a nylon membrane with theseparated DNA fragments in the 4% Nuseive 3:1 agarose gel to the nylonmembrane. Followed by lightly washing the blotted filter with 2×SSCbuffer (0.3M NaCl, 0.33M Na-Citrate, pH 7.0), the blotted nylon membraneis dried at 80° C. for 90 minutes.

The blotted nylon membrane is treated at 55° C. for 16 hours withprehybridization buffer (6×SSPE (0.9M NaCl, 0.052M NaH₂PO₄, 7.5 mMEDTA), 0.5% SDS, 5×Denhart and 0.1 mg/ml of salmon sperm DNA). Theprehybridization buffer is then exchanged with an equal volume ofhybridization buffer (6×SSPE (0.9M NaCl, 0.052M NaH₂PO₄, 7.5 mM EDTA),0.5% SDS, 5×Denhart, 0.1 mg/ml of salmon sperm DNA and a ³²P labeledprobe oligonucleotide). In the hybridization buffer, the radioactiveconcentration of the ³²P labeled probe oligonucleotide is at least10×10⁸ cpm for every 150 ml of the hybridization buffer. As the ³²Plabeled probe oligonucleotide, there is utilized the oligonucleotidedepicted in SEQ ID:81 which is labeled with ³²P at the ends thereof. The³²P labeled probe is produced by incubating at 37° C. for 1 hour with γ³²P-ATP, T4 polynucleotide kinase and 1 μg of the oligonucleotidedepicted in SEQ ID:81 in the buffer provided with the T4 polynucleotidekinase.

After the hybridization, the blotted nylon membrane is washed twice withwashing buffer containing 1×SSC (0.15 M NaCl, 15 mM sodium citrate) and0.5% SDS. In washing the blotting filter twice, the blotted nylonmembrane is incubated after each washing at 62° C. for 40 minutes in thewashing buffer.

The blotted membrane is then autoradiographed for 10 days with x-rayfilm to analyze whether the restriction enzyme Stu I is successful inrestriction digesting at the restriction site therein overlapping withthe codon in the searching region which is suspected to be a variantcodon encoding a substituted amino acid at relative position 494. Asuccessful restriction digest with the restriction enzyme Stu Iindicates that there is in the test ERα polynucleotide, a nucleotidesequence encompassing AGGCCT, overlapping with the codon encoding theamino acid at relative position 494. In such cases, it is determinedthat the test ERα is a normal ERα. An unsuccessful restriction digestwith the restriction enzyme Stu I at the corresponding locus, indicatesthat there is in the test ERα polynucleotide, a variant codon encoding asubstituted amino acid at relative position 494. In such cases, thesearching region is sequenced with an ABI autosequencer (Model 377,Applied Biosystems), to determine the mutation in the variant codon, ifpresent, in the searching region.

6.12. Example 12 Production of a Plasmid Encoding Human Normal AR

A human prostate cDNA library (CLONETECH, Quick clone cDNA#7123-1) isutilized to PCR amplify therefrom a cDNA encoding a human normal AR(Genbank Accession No. M23263). The PCR mixture in this PCRamplification contains 10 ng of the human prostate cDNA library, 10 pmolof an oligonucleotide depicted in SEQ ID:176, 10 pmol of anoligonucleotide depicted in SEQ ID:177, LA-Taq Polymerase (TakaraShuzo), the buffer provided with the LA-Taq Polymerase and dNTPs (dATP,dTTP, dGTP, dCTP). The oligonucleotides depicted in SEQ ID:176 and SEQID:177 are synthesized with a DNA synthesizer (Model 394, AppliedBiosystems,). In this PCR amplification, there is repeated 35 times witha PCRsystem 9700 (Applied Biosystems), an incubation cycle entailing anincubation at 95° C. for 1 minute followed by an incubation at 68° C.for 3 minutes.

The resulting PCR mixture is subjected to low melting point agarose gelelectrophoresis (Agarose L: Nippon Gene) to confirm with ethidiumbromide staining, that the cDNA encoding the human normal AR is PCRamplified. After recovering the amplified cDNA from the low meltingpoint agarose gel, a sample of the recovered cDNA is prepared with a DyeTerminator Sequence Kit FS (Applied Biosystems). The prepared sample ofthe cDNA is sequenced with an ABI autosequencer (Model 377, AppliedBiosystems).

Another PCR amplification is then conducted to add a Kozak consensussequence immediately upstream from the stair codon (ATG) in the cDNA.The PCR mixture in this PCR amplification contains 100 ng of the cDNAencoding the human normal AR, an oligonucleotide depicted in SEQ ID:178and an oligonucleotide depicted in SEQ ID:179, LA-Taq Polymerase (TakaraShuzo), the buffer provided with the LA-Taq Polymerase and dNTPs (dATP,dTTP, dGTP, dCTP). In this PCR amplification, there is repeated 25 timesan incubation cycle entailing an incubation at 95° C. for 1 minutefollowed by an incubation at 68° C. for 3 minutes. The resulting PCRmixture is subjected to low melting point agarose gel electrophoresis(Agarose L: Nippon Gene). After recovering the amplified cDNA from thelow melting point agarose gel, 1 μg of the amplified cDNA is treatedwith a DNA Blunting Kit (Takara Shuzo) to blunt the ends of theamplified cDNA. Subsequently, the resulting cDNA therefrom is allowed toreact with a T4 polynucleotide kinase to phosphorylate the ends thereof.After phenol treating the phosphorylated cDNA, the phosphorylated cDNAis ethanol precipitated to achieve a purified form of the phosphorylatedcDNA.

The plasmid pRc/RSV (Invitrogen) is restriction digested withrestriction enzyme Hind III and is then treated with BAP for 1 hour at65° C. The restriction digested pRc/RSV is then purified by a phenoltreatment and ethanol precipitation. The restriction digested pRc/RSV isthen treated with a DNA Blunting Kit (Takara Shuzo) to blunt the endsthereof and is subjected to low melting point agarose gelelectrophoresis (Agarose L, Nippon Gene). After recovering therestriction digested pRc/RSV from the low melting point agarose gel, 100ng of the restriction digested pRc/RSV and all of the above purifiedform of the phosphorylated cDNA are used in a ligation reaction with aT4 DNA ligase. The ligation reaction mixture is used to transform E.coli competent DH5α cells (TOYOBO). The transformed E. coli cells arecultured in LB-amp. The clones thereof showing an ampicillin resistanceare then recovered. Some of the clones are then used to isolatetherefrom the plasmids derived from the ligation reaction. An aliquotsample of each of the isolated plasmids is then prepared with a DyeTerminator Sequence Kit FS (Applied Biosystems). The isolated plasmidsare sequenced with an ABI autosequencer (Model 377, Applied Biosystems),to confirm that there is a plasmid encoding the human normal AR. Such aplasmid is selected and is designated as pRc/RSV-hAR Kozak.

6.13. Example 13 Production of a Plasmid Encoding a Human Normal GR

A human liver cDNA library (CLONETECH, Quick clone cDNA#7113-1) isutilized to PCR amplify therefrom a cDNA encoding a normal GR (GenbankAccession No. M10901). The PCR mixture in this PCR amplificationcontains 10 ng of the human liver cDNA library, 10 pmol of anoligonucleotide depicted in SEQ ID:180, 10 pmol of an oligonucleotidedepicted in SEQ ID:181, LA-Taq Polymerase (Takara Shuzo), the bufferprovided with the LA-Taq Polymerase and dNTPs (dATP, dTTP, dGTP, dCTP).The oligonucleotides depicted in SEQ ID:180 and SEQ ID:181 aresynthesized with a DNA synthesizer (Model 394, Applied Biosystems). Inthis PCR amplification, there is repeated 35 times with a PCRsystem 9700(Applied Biosystems), an incubation cycle entailing an incubation at 95°C. for 1 minute followed by an incubation at 60° C. for 3 minutes.

The resulting PCR mixture is subjected to low melting point agarose gelelectrophoresis (Agarose L: Nippon Gene) to confirm with ethidiumbromide staining, that the cDNA encoding the human normal GR is PCRamplified. After recovering the amplified cDNA from the low meltingpoint agarose gel, a sample of the recovered cDNA is prepared with a DyeTerminator Sequence Kit FS (Applied Biosystems). The prepared sample ofthe cDNA is sequenced with an ABI autosequencer (Model 377, AppliedBiosystems).

Another PCR amplification is then conducted to add a Kozak consensussequence immediately upstream from the start codon (ATG) in the cDNA.The PCR mixture in this PCR amplification contains 100 ng of the cDNAencoding the human normal GR, an oligonucleotide depicted in SEQ ID:182and an oligonucleotide depicted in SEQ ID:183, LA-Taq Polymerase (TakaraShuzo), the buffer provided with the LA-Taq Polymerase and dNTPs (dATP,dTTP, dGTP, dCTP). In this PCR amplification, there is repeated 25 timesan incubation cycle entailing an incubation at 95° C. for 1 minutefollowed by an incubation at 60° C. for 3 minutes. The resulting PCRmixture is subjected to low melting point agarose gel electrophoresis(Agarose L: Nippon Gene). After recovering the amplified cDNA from thelow melting point agarose gel, 1 μg of the amplified cDNA is treatedwith a DNA Blunting Kit (Takara Shuzo) to blunt the ends of theamplified cDNA. Subsequently, the resulting cDNA therefrom is allowed toreact with a T4 polynucleotide kinase to phosphorylate the ends of thecDNA. After phenol treating the phosphorylated cDNA, the phosphorylatedcDNA is ethanol precipitated to achieve a purified form of thephosphorylated cDNA.

The plasmid pRc/RSV (Invitrogen) is restriction digested withrestriction enzyme Hind III and is then treated with BAP for 1 hour at65° C. The restriction digested pRc/RSV is then purified by a phenoltreatment and ethanol precipitation. The restriction digested pRc/RSV istreated with a DNA Blunting Kit (Takara Shuzo) to blunt the ends thereofand is subjected to low melting point agarose gel electrophoresis(Agarose L, Nippon Gene). After recovering the restriction digestedpRc/RSV from the low melting point agarose gel, 100 ng of therestriction digested pRc/RSV and all of the above purified form of thephosphorylated cDNA are used in a ligation reaction with a T4 DNAligase. The reaction mixture is used to transform E. coli competent DH5αcells (TOYOBO). The transformed E. coli cells are cultured in LB-amp.The clones thereof showing an ampicillin resistance are then recovered.Some of the clones are then used to isolate therefrom the plasmidsderived from the ligation reaction. An aliquot sample of each of theisolated plasmids are then prepared with a Dye Terminator Sequence KitFS (Applied Biosystems). The isolated plasmids are sequenced with an ABIautosequencer (Model 377, Applied Biosystems), to confirm that there isa plasmid encoding the normal GR. The plasmid is selected and isdesignated as pRc/RSV-hGR Kozak.

6.14. Example 14 Production of a Plasmid Encoding a Human Normal PR

A human liver cDNA library (CLONETECH, Quick clone cDNA#7113-1) isutilized to PCR amplify therefrom a cDNA encoding a normal PR (GenbankAccession No. M15716). The PCR mixture in this PCR amplificationcontains 10 ng of the human liver cDNA library, 10 pmol of aoligonucleotide depicted in SEQ ID:184, 10 pmol of a oligonucleotidedepicted in SEQ ID:185, LA-Taq Polymerase (Takara Shuzo), the bufferprovided with the LA-Taq Polymerase and dNTPs (dATP, dTTP, dGTP, dCTP).The oligonucleotides depicted in SEQ ID:184 and SEQ ID:185 aresynthesized with a DNA synthesizer (Model 394, Applied Biosystems). Inthis PCR amplification, there is repeated 35 times with a PCRsystem 9700(Applied Biosystems), an incubation cycle entailing an incubation at 95°C. for 1 minute followed by an incubation at 55° C. for 3 minutes.

The resulting PCR mixture is subjected to low melting point agarose gelelectrophoresis (Agarose L: Nippon Gene) to confirm with ethidiumbromide staining, that the cDNA encoding human normal PR is PCRamplified. After recovering the amplified cDNA from the low meltingpoint agarose gel, a sample of the recovered cDNA is prepared with a DyeTerminator Sequence Kit FS (Applied Biosystems). The prepared sample ofthe cDNA is sequenced with an ABI autosequencer (Model 377, AppliedBiosystems).

Another PCR amplification is then conducted to add a Kozak consensussequence immediately upstream from the start codon (ATG) in the cDNA.The PCR mixture in this PCR amplification contains 100 ng of the cDNAencoding normal PR, a oligonucleotide depicted in SEQ ID:186 and aoligonucleotide depicted in SEQ ID:187, LA-Taq Polymerase (TakaraShuzo), the buffer provided with the LA-Taq Polymerase and dNTPs (dATP,dTTP, dGTP, dCTP). In this PCR amplification, there is repeated 25 timesan incubation cycle entailing an incubation at 95° C. for 1 minutefollowed by an incubation at 55° C. for 3 minutes. The resulting PCRmixture is subjected to low melting point agarose gel electrophoresis(Agarose L: Nippon Gene). After recovering the amplified test cDNA fromthe low melting point agarose gel, 1 μg of the amplified test cDNA istreated with a DNA Blunting Kit (Takara Shuzo) to blunt the ends of theamplified test cDNA. Subsequently, the resulting test cDNA therefrom isallowed to react with a T4 polynucleotide kinase to phosphorylate theends of the cDNA. After phenol treating the phosphorylated test cDNA,the phosphorylated test cDNA is ethanol precipitated to achieve apurified form of the phosphorylated test cDNA.

The plasmid pRc/RSV (Invitrogen) is restriction digested withrestriction enzyme Hind III and is then treated with BAP for 1 hour at65° C. The restriction digested pRc/RSV is then purified by a phenoltreatment and ethanol precipitation. The restriction digested pRc/RSV istreated with a DNA Blunting Kit (Takara Shuzo) to blunt the ends thereofand is subjected to low melting point agarose gel electrophoresis(Agarose L, Nippon Gene). After recovering the restriction digestedpRc/RSV from the low melting point agarose gel, 100 ng of therestriction digested pRc/RSV and all of the above purified form of thephosphorylated test cDNA are used in a ligation reaction with a T4 DNAligase. The ligation reaction mixture is used to transform E. colicompetent DH5α cells (TOYOBO). The transformed E. coli cells arecultured in LB-amp. The clones thereof showing an ampicillin resistanceare then recovered. Some of the clones are then used to isolatetherefrom the plasmids derived from the ligation reaction. An aliquotsample of each of the isolated plasmids are then prepared with a DyeTerminator Sequence Kit FS (Applied Biosystems). The isolated plasmidsare sequenced with an ABI autosequencer (Model 377, Applied Biosystems),to confirm that there is a plasmid encoding normal PR. Such a plasmid isselected and is designated as pRc/RSV-hPR Kozak.

6.15. Example 15 Production of a Plasmid Encoding a Human Normal MR

A human liver cDNA library (CLONETECH, Quick clone cDNA#7113-1) isutilized to PCR amplify therefrom a cDNA encoding a normal MR (GenbankAccession No. M16801). The PCR mixture in this PCR amplificationcontains 10 ng of the human liver cDNA library, 10 pmol of anoligonucleotide depicted in SEQ ID:188, 10 pmol of an oligonucleotidedepicted in SEQ ID:189, LA-Taq Polymerase (Takara Shuzo), the bufferprovided with the LA-Taq Polymerase and dNTPs (dATP, dTTP, dGTP, dCTP).The oligonucleotides depicted in SEQ ID:188 and SEQ ID:189 aresynthesized with a DNA synthesizer (Model 394, Applied Biosystems). Inthis PCR amplification, there is repeated 35 times with a PCRsystem 9700(Applied Biosystems), an incubation cycle entailing an incubation at 95°C. for 1 minute followed by an incubation at 60° C. for 3 minutes.

The resulting PCR mixture is subjected to low melting point agarose gelelectrophoresis (Agarose L: Nippon Gene) to confirm with ethidiumbromide staining, that the cDNA encoding the normal MR is PCR amplified.After recovering the amplified cDNA from the low melting point agarosegel, a sample of the recovered cDNA is prepared with a Dye TerminatorSequence Kit FS (Applied Biosystems). The prepared sample of the cDNA issequenced with an ABI autosequencer (Model 377, Applied Biosystems).

Another PCR amplification is then conducted to add a Kozak consensussequence immediately upstream from the start codon (ATG) in the cDNA.The PCR mixture in this PCR amplification contains 100 ng of the cDNAencoding the normal MR, a oligonucleotide depicted in SEQ ID:190 and aoligonucleotide depicted in SEQ ID:191, LA-Taq Polymerase (TakaraShuzo), the buffer provided with the LA-Taq Polymerase and dNTPs (dATP,dTTP, dGTP, dCTP). In this PCR amplification, there is repeated 25 timesan incubation cycle entailing an incubation at 95° C. for 1 minutefollowed by an incubation at 60° C. for 3 minutes. The resulting PCRmixture is subjected to low melting point agarose gel electrophoresis(Agarose L: Nippon Gene). After recovering the amplified cDNA from thelow melting point agarose gel, 1 μg of the amplified cDNA is treatedwith a DNA Blunting Kit (Takara Shuzo) to blunt the ends of theamplified cDNA. Subsequently, the resulting cDNA therefrom is allowed toreact with a T4 polynucleotide kinase to phosphorylate the ends of thecDNA. After phenol treating the phosphorylated cDNA, the phosphorylatedcDNA is ethanol precipitated to achieve a purified form of thephosphorylated cDNA.

The plasmid pRc/RSV (Invitrogen) is restriction digested withrestriction enzyme Hind III and is then treated with BAP for 1 hour at65° C. The restriction digested pRc/RSV is then purified by a phenoltreatment and ethanol precipitation. The restriction digested pRc/RSV istreated with a DNA Blunting Kit (Takara Shuzo) to blunt the ends thereofand is subjected to low melting point agarose gel electrophoresis(Agarose L, Nippon Gene). After recovering the restriction digestedpRc/RSV from the low melting point agarose gel, 100 ng of therestriction digested pRc/RSV and all of the above purified form of thephosphorylated cDNA are used in a ligation reaction with a T4 DNAligase. The ligation reaction mixture is used to transform E. colicompetent DH5α cells (TOYOBO). The transformed E. coli cells arecultured in LB-amp. The clones thereof showing an ampicillin resistanceare then recovered. Some of the clones are then used to isolatetherefrom the plasmids derived from the ligation reaction. An aliquotsample of each of the isolated plasmids are then prepared with a DyeTerminator Sequence Kit FS (Applied Biosystems). The isolated plasmidsare sequenced with an ABI autosequencer (Model 377, Applied Biosystems),to confirm that there is a plasmid encoding normal MR. The plasmid isselected and is designated as pRc/RSV-hMR Kozak.

6.16. Example 16 Production of a Stably Transformed Cell which StablyContains in One of its Chromosomes the MMTV Reporter Gene

The plasmid pMSG (Pharmacia) is restriction digested with restrictionenzymes Hind III and Sma I to provide a DNA fragment encoding a partialsequence of the MMTV-LTR region, which has a size of 1463 bp. The 1463bp DNA fragment is then treated with a DNA Blunting Kit (Takara Shuzo)to blunt the ends of the 1463 bp DNA fragment.

The plasmid pGL3 (Promega), which encodes the firefly luciferase gene,is restriction digested with restriction enzymes Bgl II and Hind III andis then treated with BAP at 65° C. for 1 hour. The restriction digestionreaction mixture is then subjected to low melting point agarose gelelectrophoresis (Agarose L, Nippon Gene) to confirm that there is a DNAfragment having a nucleotide sequence encoding the firefly luciferase.The DNA fragment having the nucleotide sequence encoding the fireflyluciferase is then recovered from the low melting point agarose gel.Subsequently, 100 ng of the recovered DNA fragment have the nucleotidesequence encoding firefly luciferase and 1 μg of the 1463 bp DNAfragment are used in a ligation reaction with T4 DNA ligase. Theligation reaction mixture is then used to transform E. coli competentDH5α cells (TOYOBO). The transformed E. coli cells are cultured inLB-amp. The clones thereof showing an ampicillin resistance are thenrecovered. Some of the clones are then used to isolate therefrom theplasmids derived from the ligation reaction. An aliquot sample of eachof the isolated plasmids are then restriction digested with restrictionenzymes Kpn I and Cla I. The restriction digestion reaction mixtures aresubjected to agarose gel electrophoresis to confirm that there is aplasmid which contains 1 copy of the 1463 bp DNA fragment operablyupstream from the DNA fragment have the nucleotide sequence encodingfirefly luciferase (hereinafter referred to as the MMTV reporter gene).Such a plasmid is selected and is designated as pGL3-MMTV.

The plasmid pUCSV-BSD (Funakoshi) is restriction digested withrestriction enzyme BamH I to prepare a DNA encoding a blasticidin Sdeaminase gene expression cassette. Further, the plasmid pGL3-MMTV isrestriction digested with restriction enzyme BamH I and is then treatedwith BAP at 65° C. for 1 hour. The resulting DNA encoding theblasticidin S deaminase gene expression cassette and the restrictiondigested pGL3-MMTV are mixed together to be used in a ligation reactionwith T4 DNA ligase. The ligation reaction mixture is used to transformE. coli competent DH5α cells. The transformed E. coli cells are culturedin LB-amp. The clones thereof showing an ampicillin resistance are thenrecovered. Some of the clones are then used to isolate therefrom theplasmids derived from the ligation reaction. An aliquot sample of eachof the isolated plasmids are then prepared with a Dye TerminatorSequence Kit FS (Applied Biosystems). The isolated plasmids aresequenced with an ABI autosequencer (Model 377, Applied Biosystems) toconfirm there is a plasmid which has a structure in which the DNAencoding a blasticidin S Deaminase gene expression cassette has beeninserted into the Bam HI restriction site in pGL3-MMTV. Such a plasmidis selected and is designated as pGL3-MMTV-BSD.

In order to produce stably transformed cells which stably contain in oneof its chromosomes the MMTV reporter gene (hereinafter referred to asthe stably transformed MMTV cassette cell), the plasmid pGL3-MMTV-BSD islinearized and introduced into HeLa cells.

The plasmid pGL3-MMTV-BSD is restriction digested with restrictionenzyme Sal I to linearize pGL3-MMTV-BSD.

Approximately 5×10⁵ HeLa cells were cultured as host cells for 1 dayusing dishes having a diameter of about 10 cm (Falcon) in DMEM medium(Nissui Pharmaceutical Co.) containing 10% FBS at 37° C. under thepresence of 5% CO₂.

The linearized pGL3-MMTV-BSD is then introduced to the cultured HeLacells by a lipofection method using lipofectamine (Life Technologies).According with the manual provided with the lipofectamine, theconditions under the lipofection method included 5 hours of treatment, 7μg/dish of the linearized pGL3-MMTV-BSD and 21 μl/dish of lipofectamine.

After the lipofection treatment, the DMEM medium is exchanged with DMEMmedium containing 10% FBS and the transformed HeLa cells are culturedfor about 36 hours. Next, the transformed HeLa cells are removed andcollected from the dish by trypsin treatment and are transferred into acontainer containing a medium to which blasticidin S is added to aconcentration of 16 μg/ml. The transformed HeLa cells are cultured insuch medium containing blasticidin S for 1 month while exchanging themedium every 3 or 4 days to a fresh batch of the medium containingblasticidin S.

The resulting clones, which are able to proliferate and produce a colonyhaving a diameter of from 1 to several mm, are transferred as a whole tothe wells of a 96-well ViewPlate (Berthold) to which medium ispreviously dispensed thereto. The colonies of the clones are furthercultured. When the clones proliferated to such a degree that theycovered 50% or more of the bottom surface of each of the wells (about 5days after the transfer), the clones are removed and collected bytrypsin treatment. The clones then are divided into 2 subcultures. Oneof the subcultures is transferred to a 96-well ViewPlate, which isdesignated as the master plate. The other subculture is transferred to a96-well ViewPlate, which is designated as the assay plate. The masterplate and the assay plate contain medium so that the clones can becultured. The master pate is continuously cultured under similarconditions.

The medium is then removed from the wells of the assay plate and theclones attached to the well walls are washed twice with PBS(−). A 5-folddiluted lysis buffer PGC50 (Toyo Ink) is added, respectively, to theclones in the wells of the assay plate at 20 μl per well. The assayplate is left standing at room temperature for 30 minutes and is set ona luminometer LB96P (Berthold), which is equipped with an automaticsubstrate injector. Subsequently, 50 μl of the substrate solution PGL100(Toyo Ink) is automatically dispensed to the lysed clones in the assayplate to measure the luciferase activity therein with the luminometerLB96P. A plurality of the clones, which exhibited a high luciferaseactivity are selected therefrom.

Samples of the selected clones are then cultured at 37° C. for 1 to 2weeks in the presence of 5% CO₂ using dishes having a diameter of about10 cm (Falcon) in charcoal dextran FBS/E-MEM medium.

The plasmid pRc/RSV-hAR Kozak is then introduced to the samples of theselected clones by a lipofection method using lipofectamine (LifeTechnologies) to provide a second round of clones. According with themanual provided with the lipofectamine, the conditions under thelipofection method included 5 hours of treatment, 7 μg/dish of theplasmids above and 21 μl/dish of lipofectamine. A DMSO solutioncontaining dihydrotestosterone (DHT), which is the natural cognateligand of a normal AR, is then added to the resulting second clones sothat the concentration of DHT in the medium is 10 nM. After culturingthe second clones for 2 days, the luciferase activity is measured,similarly to the above, for each of the second clones. The clone in themaster plate, which provided the second clone exhibiting the highestinduction of luciferase activity, is selected as the stably transformedMMTV cassette cell.

In this regard, the stably transformed MMTV cassette cell can be used inreporter assays with AR, GR, PR, MR and the like.

6.17. Example 17 Reporter Assay of the Human Normal AR as a Human TestAR 6.17.1. Preparation of Stably Transformed MMTV Cassette Cell

Approximately 2×10⁶ stably transformed MMTV cassette cells provided in6.16., are cultured at 37° C. for 1 day in the presence of 5% CO₂ usingdishes having a diameter of about 10 cm (Falcon) in charcoal dextranFBS/E-MEM medium.

For transient expression, the plasmid pRc/RSV-hAR Kozak is introducedinto a subculture of the stably transformed MMTV cassette cells by alipofection method using lipofectamine (Life Technologies). Accordingwith the manual provided with the lipofectamine, the conditions underthe lipofection method include 5 hours of treatment, 7 μg/dish of thepRc/RSV-hAR Kozak and 21 μl/dish of lipofectamine. After culturing theresulting cell subculture at 37° C. for 16 hours in the presence of 5%CO₂, the charcoal dextran FBS/E-MEM medium therein is exchanged to freshbatches of the charcoal dextran FBS/E-MEM medium to further culture thecell subculture for 3 hours. The cell subculture is then collected anduniformly suspended in charcoal dextran FBS/E-MEM medium to provide asubculture thereof.

6.17.2. Measurement of the Activity for Transactivation of the MMTVReporter Gene

First DMSO solutions are prepared to contain various concentrations offlutamide. The flutamide is used in the first DMSO solution as anagonist directed to the normal AR. Further, second DMSO solutions areprepared to contain 10 nM of DHT and the various concentrations of theflutamide. The flutamide is used in the second DMSO solution as anantagonist directed to the normal AR.

The first and second DMSO solutions are then mixed, respectively, withthe subcultures prepared in the above 6.17.1., in the 96-well ViewPlatessuch that the concentration of the first or second DMSO solution in eachof the wells is about 0.1% (v/v). Further, as a standard, a sample ofthe cell subculture which is provided in 6.17.1., is nixed with a DMSOsolution containing DHT, in the wells of a 96-ViewPlate.

The cells are then cultured for 40 hours at 37° C. in the presence of 5%CO₂. A 5-fold diluted lysis buffer PGC50 (Toyo Ink) is added,respectively, to the subcultures in the wells at 50 μl per well. The96-well ViewPlates are periodically and gently shook while beingincubated at room temperature for 30 minutes. Ten microliters (10 μl) ofthe lysed cells are then transferred, respectively, to white 96-wellsample plates (Berthold) and are set on a luminometer LB96P (Berthold),which is equipped with an automatic substrate injector. Subsequently, 50μl of the substrate solution PGL100 (Toyo Ink) is automaticallydispensed, respectively, to each of the lysed cells in the white 96-wellsample plates to instantaneously measure for 5 seconds the luciferaseactivity therein with the luminometer LB96P.

Further, the above reporter assay can use as the test AR, a mutant AR.In this regard, a plasmid encoding a mutant AR is used instead ofpRc/RSV-hAR Kozak. To provide the plasmid encoding the mutant AR, aKozak consensus sequence is added operably upstream from apolynucleotide encoding a mutant AR and the resulting polynucleotide isinserted into a restriction site of Hind III in the plasmid pRc/RSV(Invitrogen), as similarly described above.

6.18. Example 18 Reporter Assay of a Human Normal GR as the Human TestGR 6.18.1. Preparation of Stably Transformed MMTV Cassette Cell

Approximately 2×10⁶ stably transformed MMTV cassette cells provided in6.16., are cultured at 37° C. for 1 day in the presence of 5% CO₂ usingdishes having a diameter of about 10 cm (Falcon) in charcoal dextranFBS/E-MEM medium.

For transient expression, the plasmid pRc/RSV-hGR Kozak is introducedinto a subculture of the stably transformed MMTV cassette cells by alipofection method using lipofectamine (Life Technologies). Accordingwith the manual provided with the lipofectamine, the conditions underthe lipofection method include 5 hours of treatment, 7 μg/dish of thepRc/RSV-hAR Kozak and 21 μl/dish of lipofectamine. After culturing theresulting cell subculture at 37° C. for 16 hours in the presence of 5%CO₂, the charcoal dextran FBS/E-MEM medium therein is exchanged to freshbatches of the charcoal dextran FBS/E-MEM medium to further culture thecell subculture for 3 hours. The cell subculture is then collected anduniformly suspended in charcoal dextran FBS/E-MEM medium.

6.18.2. Measurement of the Activity for Transactivation of the MMTVReporter Gene

First DMSO solutions are prepared to contain various concentrations ofpregnanolone 16α carbonitrile (PCN). The PCN is used in the first DMSOsolutions as an agonist with the normal GR. Further, the second DMSOsolutions are prepared to contain 10 nM of corticosterone and thevarious concentrations of PCN. The PCN is used in the second DMSOsolutions as an antagonist with the normal GR.

The first and second DMSO solutions are then mixed, respectively, withthe cell subcultures prepared in the above 6.18.1., in wells of the96-well ViewPlates such that the concentration of the first or secondDMSO solution in each of the wells is about 0.1% (v/v). Further, as astandard, a sample of the cell subculture is mixed with a DMSO solutioncontaining corticosterone in the wells of a 96-well ViewPlate.

The cells are then cultured for 40 hours at 37° C. in the presence of 5%CO₂. A 5-fold diluted lysis buffer PGC50 (Toyo Ink) is added,respectively, to the subcultures in the wells at 50 μl per well. The96-well ViewPlates are periodically and gently shook while beingincubated at room temperature for 30 minutes. Ten microliters (10 μl) ofthe lysed cells are then transferred, respectively, to white 96-wellsample plates (Berthold) and are set on a luminometer LB96P (Berthold),which is equipped with an automatic substrate injector. Subsequently, 50μl of the substrate solution PGL100 (Toyo Ink) is automaticallydispensed, respectively, to each of the lysed cells in the white 96-wellsample plates to instantaneously measure for 5 seconds the luciferaseactivity therein with the luminometer LB96P.

Further, the above reporter assay can use as the test GR, a mutant GR.In this regard, a plasmid encoding the mutant GR is used instead ofpRc/RSV-hGR Kozak. To provide the plasmid encoding the mutant GR, aKozak consensus sequence is added operably upstream from apolynucleotide encoding a mutant GR and the resulting polynucleotide isinserted into a restriction site of Hind III in the plasmid pRc/RSV(Invitrogen), as similarly described above.

6.19. Example 19 Reporter Assay of Human Normal PR as the Human Test PR6.19.1. Preparation of Stably Transformed MMTV Cassette Cell

Approximately 2×10⁶ stably transformed MMTV cells provided in 6.16., arecultured at 37° C. for 1 day in the presence of 5% CO₂ using disheshaving a diameter of about 10 cm (Falcon) in charcoal dextran FBS/E-MEMmedium.

For transient expression the plasmid pRc/RSV-hPR Kozak is introducedinto a subculture of the stably transformed MMTV cassette cells by alipofection method using lipofectamine (Life Technologies). Accordingwith the manual provided with the lipofectamine, the conditions underthe lipofection method include 5 hours of treatment, 7 μg/dish of thepRc/RSV-hAR Kozak and 21 μl/dish of lipofectamine. After culturing theresulting cell subculture at 37° C. for 16 hours in the presence of 5%CO₂, the charcoal dextran FBS/E-MEM medium therein is exchanged to freshbatches of the charcoal dextran FBS/E-MEM medium to further culture eachof the cell subculture for 3 hours. The cell subculture is thencollected and uniformly suspended in charcoal dextran FBS/E-MEM medium.

6.19.2. Measurement of the Activity for Transactivation of the MMTVReporter Gene

First DMSO solutions are prepared to contain various concentrations ofRU486. The RU486 is used in the first DMSO solutions as an agonistdirected to the normal PR. Further, second DMSO solutions are preparedto contain 10 nM of progesterone and the various concentrations ofRU486. The RU486 is used in the second DMSO solutions as an antagonistdirected to the normal PR.

The first and second DMSO solutions are mixed, respectively, with thecell subcultures prepared in the above 6.19.1., in the 96-wellViewPlates such that the concentration of the first or second DMSOsolution in each of the wells is about 0.1% (v/v). Further, as astandard, a sample of the cell subculture is mixed with a DMSO solutioncontaining progesterone.

The cells are then cultured for 40 hours at 37° C. in the presence of 5%CO₂. A 5-fold diluted lysis buffer PGC50 (Toyo Ink) is added,respectively, to the cells in the wells at 50 μl per well. The 96-wellViewPlates are periodically and gently shook while being incubated atroom temperature for 30 minutes. Ten microliters (10 μl) of the lysedcells are then transferred, respectively, to white 96-well sample plates(Berthold) and are set on a luminometer LB96P (Berthold), which isequipped with an automatic substrate injector. Subsequently, 50 μl/wellof the substrate solution PGL100 (Toyo Ink) is automatically dispensed,respectively, to each of the lysed cells in the white 96-well sampleplates to instantaneously measure for 5 seconds the luciferase activitytherein with the luminometer LB96P.

Further, the above reporter assay can use as the test PR, a mutant PR.In this regard, a plasmid encoding the mutant PR is used instead ofpRc/RSV-hPR Kozak. To provide the plasmid encoding the mutant PR, aKozak consensus sequence is added operably upstream from apolynucleotide encoding a mutant PR and the resulting polynucleotide isinserted into a restriction site of Hind III in the plasmid pRc/RSV(Invitrogen), as similarly described above.

6.20. Example 20 Production of a Plasmid Encoding Human Normal ERβ

A human prostate cDNA library (CLONETECH, Quick clone cDNA#7123-1) isutilized to PCR amplify therefrom a cDNA encoding a human normal ERβ(Genbank Accession No. AB006590). The PCR mixture in this PCRamplification contains 10 ng of the human liver cDNA library, 10 pmol ofan oligonucleotide depicted in SEQ ID:192, 10 pmol of an oligonucleotidedepicted in SEQ ID:193, LA-Taq Polymerase (Takara Shuzo), the bufferprovided with the LA-Taq Polymerase and dNTPs (dATP, dTTP, dGTP, dCTP).The oligonucleotides depicted in SEQ ID:192 and SEQ ID:193 aresynthesized with a DNA synthesizer (Model 394, Applied Biosystems). Inthis PCR amplification, there is repeated 35 times with a PCRsystem 9700(Applied Biosystems), an incubation cycle entailing an incubation at 95°C. for 1 minute followed by an incubation at 68° C. for 3 minutes.

The resulting PCR mixture is subjected to low melting point agarose gelelectrophoresis (Agarose L: Nippon Gene) to confirm with ethidiumbromide staining, that the cDNA encoding human normal ERβ is PCRamplified. After recovering the amplified cDNA from the low meltingpoint agarose gel, a sample of the recovered cDNA is prepared with a DyeTerminator Sequence Kit FS (Applied Biosystems). The prepared sample ofthe cDNA is sequenced with an ABI autosequencer (Model 377, AppliedBiosystems).

Another PCR amplification is then conducted to add a Kozak consensussequence immediately upstream from the start codon (ATG) in the cDNA.The PCR mixture in this PCR amplification contains 100 ng of the cDNA,10 pmol of an oligonucleotide depicted in SEQ ID:194 and 10 pmol of anoligonucleotide depicted in SEQ ID:195, LA-Taq Polymerase (TakaraShuzo), the buffer provided with the LA-Taq Polymerase and dNTPs (dATP,dTTP, dGTP, dCTP). In this PCR amplification, there is repeated 25 timesan incubation cycle entailing an incubation at 95° C. for 1 minutefollowed by an incubation at 68° C. for 3 minutes. The resulting PCRmixture is subjected to low melting point agarose gel electrophoresis(Agarose L: Nippon Gene). After recovering the amplified cDNA from thelow melting point agarose gel, 1 μg of the amplified cDNA is treatedwith a DNA Blunting Kit (Takara Shuzo) to blunt the ends of theamplified cDNA. Subsequently, the resulting cDNA therefrom is allowed toreact with a T4 polynucleotide kinase to phosphorylate the ends of thecDNA. After phenol treating the phosphorylated cDNA, the phosphorylatedcDNA is ethanol precipitated to achieve a purified form of thephosphorylated cDNA.

The plasmid pRc/RSV (Invitrogen) is restriction digested withrestriction enzyme Hind III and is then treated with BAP for 1 hour at65° C. The restriction digested pRc/RSV is then purified by a phenoltreatment and ethanol precipitation. The restriction digested pRc/RSV istreated with a DNA Blunting Kit (Takara Shuzo) to blunt the ends thereofand is subjected to low melting point agarose gel electrophoresis(Agarose L, Nippon Gene). After recovering the restriction digestedpRc/RSV from the low melting point agarose gel, 100 ng of therestriction digested pRc/RSV and all of the above purified form of thephosphorylated cDNA are used in a ligation reaction with a T4 DNAligase. The ligation reaction mixture is used to transform E. colicompetent DH5α cells (TOYOBO). The transformed E. coli cells arecultured in LB-amp. The clones thereof showing an ampicillin resistanceare then recovered. Some of the clones are then used to isolatetherefrom the plasmids derived from the ligation reaction. An aliquotsample of the isolated plasmids are then prepared with a Dye TerminatorSequence Kit FS (Applied Biosystems). The isolated plasmids aresequenced with an ABI autosequencer (Model 377, Applied Biosystems), toconfirm that there is a plasmid encoding human normal ERβ. Such aplasmid is selected and is designated as pRc/RSV-hERβ Kozak.

6.21. Example 21 Reporter Assay of Human Normal ERβ as the Human TestERβ 6.21.1. Preparation of Stably Transformed ERE Cassette Cell

Approximately 2×10⁶ stably transformed ERE cassette cells provided in6.3., are cultured at 37° C. for 1 day in the presence of 5% CO₂ usingdishes having a diameter of about 10 cm (Falcon) in charcoal dextranFBS/E-MEM medium.

For transient expression, the plasmid pRc/RSV-hERβ Kozak is introducedinto a subculture of the stably transformed ERE cassette cells by alipofection method using lipofectamine (Life Technologies). Accordingwith the manual provided with the lipofectamine, the conditions underthe lipofection method include 5 hours of treatment, 7 μg/dish of thepRc/RSV-hERβ Kozak and 21 μl/dish of lipofectamine. After culturing theresulting cell subculture at 37° C. for 16 hours in the presence of 5%CO₂, the charcoal dextran FBS/E-MEM medium therein is exchanged to freshbatches of the charcoal dextran FBS/E-MEM medium to further culture thecell subculture for 3 hours. The cell subculture is then collected anduniformly suspended in charcoal dextran FBS/E-MEM medium.

6.21.2. Measurement of the Activity for Transactivation of the EREReporter Gene

First DMSO solutions are prepared to contain various concentrations of4-hydroxytamoxifen. The 4-hydroxytamoxifen is used in the first DMSOsolutions as an agonist directed to the human normal ERβ. Further,second DMSO solutions are prepared to contain 10 nM of E2 and thevarious concentrations of 4-hydroxytamoxifen. The 4-hydroxytamoxifen isused in the second DMSO solutions as an antagonist directed to the ERβ.

The first and second DMSO solutions are mixed, respectively, with thesubcultures prepared in the above 6.21.1., in the 96-well ViewPlatessuch that the concentration of the first or second DMSO solution in eachof the wells is about 0.1% (v/v). Further, as a standard, a sample ofthe cells is mixed with DMSO solutions containing E2 in the wells of a96-well ViewPlate.

The cells are then cultured for 40 hours at 37° C. in the presence of 5%CO₂. A 5-fold diluted lysis buffer PGC50 (Toyo Ink) is added,respectively, to the cells in the wells at 50 μl per well. The 96-wellViewPlates are periodically and gently shook while being incubated atroom temperature for 30 minutes. Ten microliters (10 μl) of the lysedcells are then transferred, respectively, to white 96-well sample plates(Berthold) and are set on a luminometer LB96P (Berthold), which isequipped with an automatic substrate injector. Subsequently, 50 μl/wellof the substrate solution PGL100 (Toyo Ink) is automatically dispensed,respectively, to each of the lysed cells in the white 96-well sampleplates to instantaneously measure for 5 seconds the luciferase activitytherein with the luminometer LB96P.

Further, the above reporter assay can use as the test ERβ, a mutant ERβ.In this regard, a plasmid encoding the mutant ERβ is used instead ofpRc/RSV-hERβ Kozak. To provide the plasmid encoding the mutant ERβ, aKozak consensus sequence is added operably upstream from apolynucleotide encoding a mutant ERβ and the resulting polynucleotide isinserted into a restriction site of Hind III in the plasmid pRc/RSV(Invitrogen), as similarly described above.

6.22. Example 22 Production of a Plasmid Encoding a Human Normal TRα

A human liver cDNA library (CLONETECH, Quick clone cDNA#7113-1) isutilized to PCR amplify therefrom a cDNA encoding a human normal TRα(Genbank Accession No. M24748). The PCR mixture in this PCRamplification contains 10 ng of the human liver cDNA library, 10 pmol ofan oligonucleotide depicted in SEQ ID:196, 10 pmol of an oligonucleotidedepicted in SEQ ID:197, LA-Taq Polymerase (Takara Shuzo), the bufferprovided with the LA-Taq Polymerase and dNTPs (dATP, dTTP, dGTP, dCTP).The oligonucleotides depicted in SEQ ID:196 and SEQ ID:197 aresynthesized with a DNA synthesizer (Model 394, Applied Biosystems). Inthis PCR amplification, there is repeated 35 times with a PCRsystem 9700(Applied Biosystems), an incubation cycle entailing an incubation at 95°C. for 1 minute followed by an incubation at 68° C. for 3 minutes.

The resulting PCR mixture is subjected to low melting point agarose gelelectrophoresis (Agarose L: Nippon Gene) to confirm with ethidiumbromide staining, that the cDNA encoding human normal TRα is PCRamplified. After recovering the amplified cDNA from the low meltingpoint agarose gel, a sample of the recovered cDNA is prepared with a DyeTerminator Sequence Kit FS (Applied Biosystems). The prepared sample ofthe cDNA is sequenced with an ABI autosequencer (Model 377, AppliedBiosystems).

Another PCR amplification is then conducted to add a Kozak consensussequence immediately upstream from the start codon (ATG) in the cDNA.The PCR mixture in this PCR amplification contains 100 ng of the cDNAencoding human normal TRα, 10 pmol of an oligonucleotide depicted in SEQID:198 and 10 pmol of an oligonucleotide depicted in SEQ ID:199, LA-TaqPolymerase (Takara Shuzo), the buffer provided with the LA-TaqPolymerase and dNTPs (dATP, dTTP, dGTP, dCTP). In this PCRamplification, there is repeated 25 times an incubation cycle entailingan incubation at 95° C. for 1 minute followed by an incubation at 68° C.for 3 minutes. The resulting PCR mixture is subjected to low meltingpoint agarose gel electrophoresis (Agarose L: Nippon Gene). Afterrecovering the amplified cDNA from the low melting point agarose gel, 1μg of the amplified cDNA is treated with a DNA Blunting Kit (TakaraShuzo) to blunt the ends of the amplified cDNA. Subsequently, theresulting cDNA therefrom is allowed to react with a T4 polynucleotidekinase to phosphorylate the ends of the cDNA. After phenol treating thephosphorylated cDNA, the phosphorylated cDNA is ethanol precipitated toachieve a purified form of the phosphorylated cDNA.

The plasmid pRc/RSV (Invitrogen) is restriction digested withrestriction enzyme Hind III and is then treated with BAP for 1 hour at65° C. The restriction digested pRc/RSV is then purified by a phenoltreatment and ethanol precipitation. The restriction digested pRc/RSV istreated with a DNA Blunting Kit (Takara Shuzo) to blunt the ends thereofand is subjected to low melting point agarose gel electrophoresis(Agarose L, Nippon Gene). After recovering the restriction digestedpRc/RSV from the low melting point agarose gel, 100 ng of therestriction digested pRc/RSV and all of the above purified form of thephosphorylated cDNA are used in a ligation reaction with a T4 DNAligase. The ligation reaction mixture is used to transform E. colicompetent DH5α cells (TOYOBO). The transformed E. coli cells arecultured in LB-amp. The clones thereof showing an ampicillin resistanceare then recovered. Some of the clones are then used to isolatetherefrom the plasmids derived from the ligation reaction. An aliquotsample of the isolated plasmids are then prepared with a Dye TerminatorSequence Kit FS (Applied Biosystems). The isolated plasmids aresequenced with an ABI autosequencer (Model 377, Applied Biosystems), toconfirm that there is a plasmid encoding human normal TRα. Such aplasmid is selected and is designated as pRc/RSV-hTRαKozak.

6.23. Example 23 Production of a Plasmid Encoding a Human Normal TRβ

A human liver cDNA library (CLONETECH, Quick clone cDNA#7113-1) isutilized to PCR amplify therefrom a cDNA encoding a human normal TRβ(Genbank Accession No. M26747). The PCR mixture in this PCRamplification contains 10 ng of the human liver cDNA library, 10 pmol ofan oligonucleotide depicted in SEQ ID:200, 10 pmol of an oligonucleotidedepicted in SEQ ID:201, LA-Taq Polymerase (Takara Shuzo), the bufferprovided with the LA-Taq Polymerase and dNTPs (dATP, dTTP, dGTP, dCTP).The oligonucleotides depicted in SEQ ID:200 and SEQ ID:201 aresynthesized with a DNA synthesizer (Model 394, Applied Biosystems). Inthis PCR amplification, there is repeated 35 times with a PCRsystem 9700(Applied Biosystems), an incubation cycle entailing an incubation at 95°C. for 1 minute followed by an incubation at 68° C. for 3 minutes.

The resulting PCR mixture is subjected to low melting point agarose gelelectrophoresis (Agarose L: Nippon Gene) to confirm with ethidiumbromide staining, that the cDNA encoding human normal TRβ is PCRamplified. After recovering the amplified cDNA from the low meltingpoint agarose gel, a sample of the recovered cDNA is prepared with a DyeTerminator Sequence Kit FS (Applied Biosystems). The prepared sample ofthe cDNA is sequenced with an ABI autosequencer (Model 377, AppliedBiosystems).

Another PCR amplification is then conducted to add a Kozak consensussequence immediately upstream from the stair codon (ATG) in the cDNA.The PCR mixture in this PCR amplification contains 100 ng of the cDNAencoding normal TRα, 10 pmol of an oligonucleotide depicted in SEQID:202 and 10 pmol of an oligonucleotide depicted in SEQ ID:203, LA-TaqPolymerase (Takara Shuzo), the buffer provided with the LA-TaqPolymerase and dNTPs (dATP, dTTP, dGTP, dCTP). In this PCRamplification, there is repeated 25 times an incubation cycle entailingan incubation at 95° C. for 1 minute followed by an incubation at 68° C.for 3 minutes. The resulting PCR mixture is subjected to low meltingpoint agarose gel electrophoresis (Agarose L, Nippon Gene). Afterrecovering the amplified cDNA from the low melting point agarose gel, 1μg of the amplified cDNA is treated with a DNA Blunting Kit (TakaraShuzo) to blunt the ends of the amplified cDNA. Subsequently, theresulting cDNA therefrom is allowed to react with a T4 polynucleotidekinase to phosphorylate the ends of the cDNA. After phenol treating thephosphorylated cDNA, the phosphorylated cDNA is ethanol precipitated toachieve a purified form of the phosphorylated cDNA.

The plasmid pRc/RSV (Invitrogen) is restriction digested withrestriction enzyme Hind III and is then treated with BAP for 1 hour at65° C. The restriction digested pRc/RSV is then purified by a phenoltreatment and ethanol precipitation. The restriction digested pRc/RSV istreated with a DNA Blunting Kit (Takara Shuzo) to blunt the ends thereofand is subjected to low melting point agarose gel electrophoresis(Agarose L, Nippon Gene). After recovering the restriction digestedpRc/RSV from the low melting point agarose gel, 100 ng of therestriction digested pRc/RSV and all of the above purified form of thephosphorylated cDNA are used in a ligation reaction with a T4 DNAligase. The ligation reaction mixture is used to transform E. colicompetent DH5α cells (TOYOBO). The transformed E. coli cells arecultured in LB-amp. The clones thereof showing an ampicillin resistanceare then recovered. Some of the clones are then used to isolatetherefrom the plasmids derived from the ligation reaction. An aliquotsample of each of the isolated plasmids are then prepared with a DyeTerminator Sequence Kit FS (Applied Biosystems). The isolated plasmidsare sequenced with an ABI autosequencer (Model 377, Applied Biosystems),to confirm that there is a plasmid encoding human normal TRβ. Such aplasmid is selected and is designated as pRc/RSV-hTRβ Kozak.

6.24. Example 24 Production of a Plasmid Containing an DR4 Reporter Gene

An oligonucleotide depicted in SEQ ID:204 and an oligonucleotide havinga nucleotide sequence complementary thereto are synthesized with a DNAsynthesizer. The oligonucleotide depicted in SEQ ID: 204 is synthesizedto encode one of the strands of an DR4. The second oligonucleotide issynthesized to have a nucleotide sequence complementary to the firstoligonucleotide. The two oligonucleotides are annealed together toproduce a DNA encoding a DR4 sequence (hereinafter referred to as theDR4 DNA). A T4 polynucleotide kinase is allowed to react with the DR4DNA to phosphorylate the ends thereof. The DR4 DNA is then ligatedtogether with a T4 DNA ligase to provide a DR4x5 DNA having a 5 tandemrepeat of the DR4 sequence. The ligation reaction mixture is thensubjected to low melting point agarose gel electrophoresis (Agarose L,Nippon Gene), and the DR4x5 DNA is recovered from the gel.

The plasmid pGL3-TATA provided in 6.2., is restriction digested withrestriction enzyme Sma I and is then treated with BAP at 65° C. for 1hour. The restriction digested reaction mixture is then subjected to lowmelting point agarose gel electrophoresis (Agarose L, Nippon Gene).After recovering the DNA fragment having a nucleotide sequence encodingfirefly luciferase from the low melting point agarose gel, 100 ng of therecovered DNA fragment and 1 μg of the DR4x5 DNA are used in a ligationreaction. The resulting ligation reaction mixture is then used totransform E. coli competent DH5α cells (TOYOBO). The transformed E. colicells are cultured in LB-amp. The clones thereof showing an ampicillinresistance are then recovered. Some of the clones are then used toisolate therefrom the plasmids derived from the ligation reaction. Analiquot sample of each of the isolated plasmids are then restrictiondigested with restriction enzymes Kpn I and Xho I. The restrictiondigestion reaction mixtures are subjected to agarose gel electrophoresisto confirm that there is a plasmid having a structure in which the DR4x5DNA is inserted into the restriction site of restriction enzyme Sma I inthe pGL3-TATA. Such a plasmid is selected and is designated aspGL3-TATA-DR4x5.

The plasmid pGL3-TATA-DR4x5 is then restriction digested withrestriction enzyme Sal I. After a Blunting Kit (Takara Shuzo) is used toblunt the ends of the restriction digested pGL3-TATA-DR4x5, therestriction digested pGL3-TATA-DR4x5 is treated with BAP at 65° C. for 1hour. The Blunting Kit is also used to blunt the ends of the DNAfragment encoding the blasticidin S deaminase gene (BamH I-BamH Ifragment) provided in 6.2.

The DNA fragment encoding a blasticidin S deaminase gene expressioncassette and the restriction digested pGL3-TATA-DR4x5 are then mixedtogether for a ligation reaction with T4 DNA ligase. The ligationreaction mixture is used to transform E. coli competent DH5α cells(TOYOBO). The transformed E. coli cells are cultured in LB-amp. Theclones thereof showing an ampicillin resistance are then recovered. Someof the clones are then used to isolate therefrom the plasmids derivedfrom the ligation reaction. An aliquot sample of each of the isolatedplasmids are then prepared with a Dye Terminator Sequence Kit FS(Applied Biosystems). The isolated plasmids are sequenced with an ABIautosequencer (Model 377, Applied Biosystems) to confirm whether thereis a plasmid which has structure in which the DNA encoding a blasticidinS deaminase gene expression cassette is inserted into the restrictionsite of restriction enzyme Sal I in pGL3-TATA-DR4x5. Such a plasmid isselected and is designated as pGL3-TATA-DR4x5-BSD.

6.25. Example 25 Production of a Stably Transformed Cell which StablyContains in One of its Chromosomes the DR4 Reporter Gene

In order to produce stably transformed cells which stably contains inone of its chromosomes the DR4 reporter gene (hereinafter referred to asthe stably transformed DR4 cassette cell, the plasmidpGL3-TATA-DR4x5-BSD was linearized and introduced into HeLa cells.

The plasmid pGL3-TATA-DR4x5-BSD is restriction digested with restrictionenzyme Not I to linearize pGL3-TATA-DR4x5-BSD.

Approximately 5×10⁵ HeLa cells are cultured as host cells for 1 dayusing dishes having a diameter of about 10 cm (Falcon) in DMEM medium(Nissui Pharmaceutical Co.) containing 10% FBS at 37° C. in the presenceof 5% CO₂.

The linearized pGL3-TATA-DR4x5-BSD is then introduced to the culturedHeLa cells by a lipofection method using lipofectamine (LifeTechnologies). According with the manual provided with thelipofectamine, the conditions under the lipofection method include 5hours of treatment, 7 μg/dish of the linearized pGL3-TATA-DR4x5-BSD and21 μl/dish of lipofectamine.

After the lipofection treatment, the DMEM medium is exchanged with DMEMmedium containing 10% FBS and the transformed HeLa cells are culturedfor about 36 hours. Next, the transformed HeLa cells are removed andcollected from the dish by trypsin treatment and are transferred into acontainer containing a medium to which blasticidin S is added to aconcentration of 16 μg/ml. The transformed HeLa cells are cultured insuch medium containing blasticidin S for 1 month while exchanging themedium containing blasticidin S every 3 or 4 days to a fresh batch ofthe medium containing blasticidin S.

The resulting clones, which are able to proliferate and produce a colonyhaving a diameter of from 1 to several mm, are transferred,respectively, as a whole to the wells of a 96-well ViewPlate (Berthold)to which medium is previously dispensed thereto. The clones are furthercultured. When the clones proliferated to such a degree that clonestherein covered 50% or more of the bottom surface of each of the wells(about 5 days after the transfer), the clones are removed and collectedby trypsin treatment. Each of the clones then are divided into 2subcultures. One of the subcultures is transferred to a 96-wellViewPlate, which is designated as the master plate. The other subculturewas transferred to a 96-well ViewPlate, which is designated as the assayplate. The master plate and the assay plate contain medium so that theclones can be cultured. The master pate is continuously cultured undersimilar conditions.

The medium in the wells of the assay plate is then removed therefrom andthe clones attached to the well walls are washed twice with PBS(−). A5-fold diluted lysis buffer PGC50 (Toyo Ink) is added, respectively, tothe clones in the wells of the assay plate at 20 μl/well. The assayplate is left standing at room temperature for 30 minutes and is set ona luminometer LB96P (Berthold), which is equipped with an automaticsubstrate injector. Subsequently, 50 μl of the substrate solution PGL100(Toyo Ink) is automatically dispensed to the lysed clones in the assayplate to measure the luciferase activity therein with the luminometerLB96P. A plurality of the clones, which exhibited a luciferase activityare selected therefrom.

Samples of the selected clones are then cultured at 37° C. for 1 day inthe presence of 5% CO₂ using dishes having a diameter of about 10 cm(Falcon) in charcoal dextran FBS/E-MEM medium.

The plasmid pRc/RSV-hTRαKozak is then introduced to the samples of theselected clones by a lipofection method using lipofectamine (LifeTechnologies) to provide a second round of clones. According with themanual provided with the lipofectamine, the conditions under thelipofection method included 5 hours of treatment, 7 μg/dish ofpRc/RSV-hTRαKozak above and 21 μl/dish of lipofectamine. A DMSO solutioncontaining triiodothyronine (T3), which is the natural cognate ligand ofa human normal TRα, is then added to the resulting second clones so thatthe concentration of T3 in the medium is 10 nM. After culturing thesecond clones for 2 days, the luciferase activity is measured, similarlyto the above, for each of the second clones. The clone in the masterplate, which provided the second clone exhibiting the highest inductionof luciferase activity, is selected as the stably transformed DR4cassette cell.

In this regard, the stably transformed DR4 cassette cell can be used inreporter assays with TRα, TRβ, CAR, LXR, PXR and the like.

6.26. Example 26 Production of a Plasmid Encoding Human Normal VDR

A human liver cDNA library (CLONETECH, Quick clone cDNA#7113-1) isutilized to PCR amplify therefrom a cDNA encoding a human normal VDR(Genbank Accession No. J03258). The PCR mixture in this PCRamplification contains 10 ng of the human liver cDNA library, 10 pmol ofan oligonucleotide depicted in SEQ ID:205, 10 pmol of an oligonucleotidedepicted in SEQ ID:206, LA-Taq Polymerase (Takara Shuzo), the bufferprovided with the LA-Taq Polymerase and dNTPs (dATP, dTTP, dGTP, dCTP).The oligonucleotides depicted in SEQ ID:205 and SEQ ID:206 aresynthesized with a DNA synthesizer (Model 394, Applied Biosystems). Inthis PCR amplification, there is repeated 35 times with a PCRsystem 9700(Applied Biosystems), an incubation cycle entailing an incubation at 95°C. for 1 minute followed by an incubation at 68° C. for 3 minutes.

The resulting PCR mixture is subjected to low melting point agarose gelelectrophoresis (Agarose L, Nippon Gene) to confirm with ethidiumbromide staining, that the cDNA encoding human normal VDR is PCRamplified. After recovering the amplified cDNA from the low meltingpoint agarose gel, a sample of the recovered cDNA is prepared with a DyeTerminator Sequence Kit FS (Applied Biosystems). The prepared sample ofthe cDNA is sequenced with an ABI autosequencer (Model 377, AppliedBiosystems).

Another PCR amplification is then conducted to add a Kozak consensussequence immediately upstream from the start codon (ATG) in the cDNAencoding normal. The PCR mixture in this PCR amplification contains 100ng of the cDNA encoding normal, 10 pmol of an oligonucleotide depictedin SEQ ID:207 and 10 pmol of an oligonucleotide depicted in SEQ ID:208,LA-Taq Polymerase (Takara Shuzo), the buffer provided with the LA-TaqPolymerase and dNTPs (dATP, dTTP, dGTP, dCTP). In this PCRamplification, there is repeated 25 times an incubation cycle entailingan incubation at 95° C. for 1 minute followed by an incubation at 68° C.for 3 minutes. The resulting PCR mixture is subjected to low meltingpoint agarose gel electrophoresis (Agarose L, Nippon Gene). Afterrecovering the amplified cDNA from the low melting point agarose gel, 1μg of the amplified cDNA is treated with a DNA Blunting Kit (TakaraShuzo) to blunt the ends of the amplified cDNA. Subsequently, theresulting cDNA therefrom is allowed to react with a T4 polynucleotidekinase to phosphorylate the ends of the cDNA. After phenol treating thephosphorylated cDNA, the phosphorylated cDNA is ethanol precipitated toachieve a purified form of the phosphorylated cDNA.

The plasmid pRc/RSV (Invitrogen) is restriction digested withrestriction enzyme Hind III and is then treated with BAP for 1 hour at65° C. The restriction digested pRc/RSV is then purified by a phenoltreatment and ethanol precipitation. The restriction digested pRc/RSV istreated with a DNA Blunting Kit (Takara Shuzo) to blunt the ends thereofand is subjected to low melting point agarose gel electrophoresis(Agarose L, Nippon Gene). After recovering the restriction digestedpRc/RSV from the low melting point agarose gel, 100 ng of therestriction digested pRc/RSV and all of the above purified form of thephosphorylated cDNA is used in a ligation reaction with a T4 DNA ligase.The ligation reaction mixture is used to transform E. coli competentDH5α cells (TOYOBO). The transformed E. coli cells are cultured inLB-amp. The clones thereof showing an ampicillin resistance are thenrecovered. Some of the clones are then used to isolate therefrom theplasmids derived from the ligation reaction. An aliquot sample of eachof the isolated plasmids are then prepared with a Dye TerminatorSequence Kit FS (Applied Biosystems). The isolated plasmids aresequenced with an ABI autosequencer (Model 377, Applied Biosystems), toconfirm that there is a plasmid encoding human normal VDR. Such aplasmid is selected and is designated as pRc/RSV-hVDR Kozak.

6.27. Example 27 Production of a Plasmid Containing a DR3 Reporter Gene

An oligonucleotide depicted in SEQ ID:209 and an oligonucleotide havinga nucleotide sequence complementary thereto are synthesized with a DNAsynthesizer. The oligonucleotide depicted in SEQ ID: 209 is synthesizedto encode one of the strands of a DR3. The second oligonucleotide issynthesized to have a nucleotide sequence complementary to the firstoligonucleotide. The two oligonucleotides are annealed together toproduce a DNA encoding a DR3 sequence (hereinafter referred to as theDR3 DNA). A T4 polynucleotide kinase is allowed to react with the DR3DNA to phosphorylate the ends thereof. The DR3 DNA is then ligatedtogether with a T4 DNA ligase to provide a DR3x5 DNA having a 5 tandemrepeat of the DR3 sequence. The ligation reaction mixture is thensubjected to low melting point agarose gel electrophoresis (Agarose L,Nippon Gene), and the DR3x5 DNA is recovered from the gel.

The plasmid pGL3-TATA provided in 6.2., is restriction digested withrestriction enzyme Sma I and is then treated with BAP at 65° C. for 1hour. The restriction digested reaction mixture is then subjected to lowmelting point agarose gel electrophoresis (Agarose L, Nippon Gene).After recovering the DNA fragment having a nucleotide sequence encodingfirefly luciferase from the low melting point agarose gel, 100 ng of therecovered DNA fragment and 1 μg of the DR3x5 DNA are used in a ligationreaction. The resulting ligation reaction mixture is then used totransform E. coli competent DH5α cells (TOYOBO). The transformed E. colicells are cultured in LB-amp. The clones thereof showing an ampicillinresistance are then recovered. Some of the clones are then used toisolate therefrom the plasmids derived from the ligation reaction. Analiquot sample of each of the isolated plasmids is then restrictiondigested with restriction enzymes Kpn I and Xho I. The restrictiondigestion reaction mixtures are subjected to agarose gel electrophoresisto confirm that there is a plasmid having a structure in which the DR3x5DNA is inserted into the restriction site of restriction enzyme Sma I inthe pGL3-TATA. Such a plasmid is selected and is designated aspGL3-TATA-DR3x5.

The plasmid pGL3-TATA-DR3x5 is then restriction digested withrestriction enzyme Sal I. After a Blunting Kit (Takara Shuzo) is used toblunt the ends of the restriction digested pGL3-TATA-DR3x5, therestriction digested pGL3-TATA-DR3x5 is treated with BAP at 65° C. for 1hour. The Blunting Kit is also used to blunt the ends of the DNAfragment having the blasticidin S deaminase gene expression cassette(BamH I-BamH I fragment) provided in 6.2. The blunt endedpGL3-TATA-DR3x5 and the blunt ended DNA fragment having the blasticidinS deaminase gene expression cassette are used in a ligation reactionwith T4 DNA ligase. The resulting ligation reaction mixture is then usedto transform E. coli competent DH5α cells (TOYOBO). The transformed E.coli cells are cultured in LB-amp. The clones thereof showing anampicillin resistance are then recovered. Some of the clones are thenused to isolate therefrom the plasmids derived from the ligationreaction. The isolated plasmid in which the DNA fragment having theblasticidin S deaminase gene expression cassette is inserted to therestriction site of restriction enzyme Sal I in pGL3-TATA-DR3x5 isselected and is designated as pGL3-TATA-DR3x5-BSD.

6.28. Example 28 Production of a Stably Transformed Cassette Cell whichStably Contains in One of its Chromosomes the DR3 Reporter Gene

In order to produce stably transformed cells which stably contain in oneof its chromosomes the DR3 reporter gene (hereinafter referred to as thestably transformed DR3 cassette cell, the plasmid pGL3-TATA-DR3x5-BSDwas linearized and introduced into HeLa cells.

The plasmid pGL3-TATA-DR3x5-BSD is restriction digested with restrictionenzyme Not I to linearize pGL3-TATA-DR3x5-BSD.

Approximately 5×10⁵ HeLa cells are cultured as host cells for 1 dayusing dishes having a diameter of about 10 cm (Falcon) in DMEM medium(Nissui Pharmaceutical Co.) containing 10% FBS at 37° C. under thepresence of 5% CO₂.

The linearized pGL3-TATA-DR3x5-BSD are then introduced to the culturedHeLa cells by a lipofection method using lipofectamine (LifeTechnologies). According with the manual provided with thelipofectamine, the conditions under the lipofection method include 5hours of treatment, 7 μg/dish of the linearized pGL3-TATA-DR3x5-BSD and21 μl/dish of lipofectamine.

After the lipofection treatment, the DMEM medium is exchanged with DMEMmedium containing 10% FBS and the transformed HeLa cells are culturedfor about 36 hours. Next, the transformed HeLa cells are removed andcollected from the dish by trypsin treatment and are transferred into acontainer containing a medium to which blasticidin S is added to aconcentration of 16 μg/ml. The transformed cells are cultured in suchmedium containing blasticidin S for 1 month while exchanging the mediumcontaining blasticidin S every 3 or 4 days to a fresh batch of the DMEMmedium containing blasticidin S.

The clones, which are able to proliferate and produce a colony having adiameter of from 1 to several mm, are transferred, respectively, as awhole to the wells of a 96-well ViewPlate (Berthold) to which medium ispreviously dispensed thereto. The clones are further cultured. When theclones proliferated to such a degree that eukaryotic clones thereincovered 50% or more of the bottom surface of each of the wells (about 5days after the transfer), the clones are removed and collected bytrypsin treatment. The clones then are divided into 2 subcultures. Oneof the subcultures is transferred to a 96-well ViewPlate, which isdesignated as the master plate. The other subculture is transferred to a96-well ViewPlate, which is designated as the assay plate. The masterplate and the assay plate contain medium so that the clones can becultured. The master pate is continuously cultured under similarconditions.

The medium in the wells of the assay plate is then removed therefrom andthe clones attached to the well walls are washed twice with PBS(−). A5-fold diluted lysis buffer PGC50 (Toyo Ink) is added, respectively, tothe clones in the wells of the assay plate at 20 μl/well. The assayplate is left standing at room temperature for 30 minutes and is set ona luminometer LB96P (Berthold), which is equipped with an automaticsubstrate injector. Subsequently, 50 μl of the substrate solution PGL100(Toyo Ink) is automatically dispensed to the lysed clones in the assayplate to measure the luciferase activity therein with the luminometerLB96P. A plurality of the clones, which exhibited a high luciferaseactivity are selected therefrom.

Samples of the selected clones are then cultured at 37° C. for 1 to 2weeks in the presence of 5% CO₂ using dishes having a diameter of about10 cm (Falcon) in charcoal dextran FBS/E-MEM medium.

The plasmid pRc/RSV-hVDR Kozak is then introduced to the samples of theselected clones by a lipofection method using lipofectamine (LifeTechnologies) to provide a second round of clones. According with themanual provided with the lipofectamine, the conditions under thelipofection method included 5 hours of treatment, 7 μg/dish ofpRc/RSV-VDR Kozak above and 21 μl/dish of lipofectamine. A DMSO solutioncontaining 1.25-(OH) Vitamin D₃, which is the natural cognate ligand ofa human normal VDR, is then added to the resulting second clones so thatthe concentration of 1.25-(OH) Vitamin D₃ in the medium is 10 nM. Afterculturing the second clones for 2 days, the luciferase activity ismeasured, similarly to the above, for each of the second clones. Theclone in the master plate, which provided the second clone exhibitingthe highest induction of luciferase activity, is selected as the stablytransformed DR3 cassette cell.

6.29. Example 29 Production of a Plasmid Encoding Normal PPAR γ

A human liver cDNA library (CLONETECH, Quick clone cDNA#7113-1) isutilized to PCR amplify therefrom a cDNA encoding a human normal PPAR γ(Genbank Accession No. U79012). The PCR mixture in this PCRamplification contains 10 ng of the human liver cDNA library, 10 pmol ofan oligonucleotide depicted in SEQ ID:210, 10 pmol of an oligonucleotidedepicted in SEQ ID:211, LA-Taq Polymerase (Takara Shuzo), the bufferprovided with the LA-Taq Polymerase and dNTPs (dATP, dTTP, dGTP, dCTP).The oligonucleotides depicted in SEQ ID:210 and SEQ ID:211 aresynthesized with a DNA synthesizer (Model 394, Applied Biosystems). Inthis PCR amplification, there is repeated 35 times with a PCRsystem 9700(Applied Biosystems), an incubation cycle entailing an incubation at 95°C. for 1 minute followed by an incubation at 68° C. for 3 minutes.

The resulting PCR mixture is subjected to low melting point agarose gelelectrophoresis (Agarose L: Nippon Gene) to confirm with ethidiumbromide staining, that the cDNA encoding human normal PPAR γ is PCRamplified. After recovering the amplified cDNA from the low meltingpoint agarose gel, a sample of the recovered cDNA is prepared with a DyeTerminator Sequence Kit FS (Applied Biosystems). The prepared sample ofthe cDNA is sequenced with an ABI autosequencer (Model 377, AppliedBiosystems).

Another PCR amplification is then conducted to add a Kozak consensussequence immediately upstream from the start codon (ATG) in the cDNA.The PCR mixture in this PCR amplification contains 100 ng of the cDNAencoding normal PPAR γ and Kozak consensus sequence, an oligonucleotidedepicted in SEQ ID:212 and an oligonucleotide depicted in SEQ ID:211,LA-Taq Polymerase (Takara Shuzo), the buffer provided with the LA-TaqPolymerase and dNTPs (dATP, dTTP, dGTP, dCTP). In this PCRamplification, there is repeated 25 times an incubation cycle entailingan incubation at 95° C. for 1 minute followed by an incubation at 68° C.for 3 minutes. The resulting PCR mixture is subjected to low meltingpoint agarose gel electrophoresis (Agarose L, Nippon Gene). Afterrecovering the amplified cDNA from the low melting point agarose gel, 1μg of the amplified cDNA is treated with a DNA Blunting Kit (TakaraShuzo) to blunt the ends of the amplified cDNA. Subsequently, theresulting cDNA therefrom is allowed to react with a T4 polynucleotidekinase to phosphorylate the ends of the cDNA. After phenol treating thephosphorylated cDNA, the phosphorylated cDNA is ethanol precipitated toachieve a purified form of the phosphorylated cDNA.

The plasmid pRc/RSV (Invitrogen) is restriction digested withrestriction enzyme Hind III and is then treated with BAP for 1 hour at65° C. The restriction digested pRc/RSV is then purified by a phenoltreatment and ethanol precipitation. The restriction digested pRc/RSV istreated with a DNA Blunting Kit (Takara Shuzo) to blunt the ends thereofand is subjected to low melting point agarose gel electrophoresis(Agarose L, Nippon Gene). After recovering the restriction digestedpRc/RSV from the low melting point agarose gel, 100 ng of therestriction digested pRc/RSV and all of the above purified form of thephosphorylated cDNA are used in a ligation reaction with a T4 DNAligase. The ligation reaction mixture is used to transform E. colicompetent DH5α cells (TOYOBO). The transformed E. coli cells arecultured in LB-amp. The clones thereof showing an ampicillin resistanceare then recovered. Some of the clones are then used to isolatetherefrom the plasmids derived from the ligation reaction. Each ofisolated plasmids is then prepared with a Dye Terminator Sequence Kit FS(Applied Biosystems). The isolated plasmids are sequenced with an ABIautosequencer (Model 377, Applied Biosystems), to confirm that there isa plasmid encoding human normal PPAR γ. Such a plasmid is selected andis designated as pRc/RSV-hPPAR γ Kozak.

6.30. Example 30 Production of a Plasmid Containing a DR1 Reporter Gene

An oligonucleotide depicted in SEQ ID:213 and an oligonucleotide havinga nucleotide sequence complementary thereto are synthesized with a DNAsynthesizer. The oligonucleotide depicted in SEQ ID: 213 is synthesizedto encode one of the strands of a DR1 sequence. The secondoligonucleotide is synthesized to have a nucleotide sequencecomplementary to the first oligonucleotide. The two oligonucleotides areannealed together to produce a DNA encoding a DR1 sequence (hereinafterreferred to as the DR1 DNA). A T4 polynucleotide kinase is allowed toreact with the DR1 DNA to phosphorylate the ends thereof. The DR1 DNA isthen ligated together with a T4 DNA ligase to provide a DR1x5 DNA havinga 5 tandem repeat of the DR 1 sequence. The ligation reaction mixture isthen subjected to low melting point agarose gel electrophoresis (AgaroseL, Nippon Gene), and the DR1x5 DNA is recovered from the gel.

The plasmid pGL3-TATA provided in 6.2., is restriction digested withrestriction enzyme Sma I and is then treated with BAP at 65° C. for 1hour. The restriction digestion reaction mixture is then subjected tolow melting point agarose gel electrophoresis (Agarose L, Nippon Gene).After recovering the DNA fragment having a nucleotide sequence encodingfirefly luciferase from the low melting point agarose gel, 100 ng of therecovered DNA fragment and 1 μg of the DR1x5 DNA are used in a ligationreaction with T4 DNA ligase. The resulting ligation reaction mixture isthen used to transform E. coli competent DH5α cells (TOYOBO). Thetransformed E. coli cells are cultured in LB-amp. The clones thereofshowing an ampicillin resistance are then recovered. Some of the clonesare then used to isolate therefrom the plasmids derived from theligation reaction. An aliquot sample of each of the isolated plasmids isthen restriction digested with restriction enzymes Kpn I and Xho I. Therestriction digestion reaction mixtures are subjected to agarose gelelectrophoresis to confirm that there is a plasmid in which the DR1x5DNA is inserted into the restriction site of restriction enzyme Sma I inthe pGL3-TATA. The plasmid is then sequenced with an ABI autosequencer(Model 377, Applied Biosystems), to confirm that there is provided aplasmid having a 5 tandem repeat of the DR1 sequence. Such a plasmid isselected and is designated as pGL3-TATA-DR1x5.

The plasmid pGL3-TATA-DR1x5 is then restriction digested withrestriction enzyme Sal I. After a Blunting Kit (Takara Shuzo) is used toblunt the ends of the restriction digested pGL3-TATA-DR1x5, therestriction digested pGL3-TATA-DR1x5 is treated with BAP at 65° C. for 1hour. The Blunting Kit is also used to blunt the ends of the DNAfragment encoding the blasticidin S deaminase gene (BamH I-BamH Ifragment derived from pUCSV-BSD (Funakoshi)) provided in 6.2.

The DNA fragment encoding a blasticidin S deaminase gene expressioncassette and the restriction digested pGL3-TATA-DR1x5 are then mixedtogether for a ligation reaction with T4 DNA ligase. The ligationreaction mixture is used to transform E. coli competent DH5α cells(TOYOBO). The transformed E. coli cells are cultured in LB-amp. Theclones thereof showing an ampicillin resistance are then recovered. Someof the clones are then used to isolate therefrom the plasmids derivedfrom the ligation reaction. An aliquot sample of each of the isolatedplasmids are then prepared with a Dye Terminator Sequence Kit FS(Applied Biosystems). The isolated plasmids are sequenced with an ABIautosequencer (Model 377, Applied Biosystems) to confirm whether theplasmid has a structure in which the DNA encoding a blasticidin Sdeaminase gene expression cassette has been inserted into therestriction site of restriction enzyme Sal I in pGL3-TATA-DR1x5. Theplasmid is selected and is designated as pGL3-TATA-DR1x5-BSD.

6.31. Example 31 Production or a Stably Transformed Cassette Cell whichStably Contain in One of its Chromosomes the DR1 Reporter Gene

In order to produce stably transformed cassette cells which stablycontain in one of its chromosomes the DR1 reporter gene (hereinafterreferred to as the stably transformed DR1 cassette cell), the plasmidpGL3-TATA-DR1x5-BSD is linearized and introduced into HeLa cells.

The plasmid pGL3-TATA-DR1x5-BSD is restriction digested with restrictionenzyme Not I to linearize pGL3-TATA-DR1x5-BSD.

Approximately 5×10⁵ HeLa cells are cultured as host cells for 1 dayusing dishes having a diameter of about 10 cm (Falcon) in DMEM medium(Nissui Pharmaceutical Co.) containing 10% FBS at 37° C. under thepresence of 5% CO₂.

The linearized pGL3-TATA-DR1x5-BSD is then introduced to the culturedHeLa cells by a lipofection method using lipofectamine (LifeTechnologies). According with the manual provided with thelipofectamine, the conditions under the lipofection method include 5hours of treatment, 7 μg/dish of the linearized pGL3-TATA-DR1x5-BSD and21 μl/dish of lipofectamine.

After the lipofection treatment, the DMEM medium is exchanged with DMEMmedium containing 10% FBS and the transformed HeLa cells are culturedfor about 36 hours. Next, the transformed HeLa cells are removed andcollected from the dish by trypsin treatment and are transferred into acontainer containing a medium to which blasticidin S is added to aconcentration of 16 μg/ml. The transformed HeLa cells are cultured insuch medium containing blasticidin S for 1 month while exchanging themedium containing blasticidin S every 3 or 4 days to a fresh batch ofthe medium containing blasticidin S.

The clones, which are able to proliferate and produce a colony having adiameter of from 1 to several mm, are transferred, respectively, as awhole to the wells of a 96-well ViewPlate (Berthold) to which medium ispreviously dispensed thereto. The clones are further cultured. When theclones proliferated to such a degree that clones therein covered 50% ormore of the bottom surface of each of the wells (about 5 days after thetransfer), the clones are removed and collected by trypsin treatment.The clones then are divided into 2 subcultures. One of the subculturesis transferred to a 96-well ViewPlate, which is designated as the masterplate. The other subculture was transferred to a 96-well ViewPlate,which is designated as the assay plate. The master plate and the assayplate contain medium so that the clones can be cultured. The master pateis continuously cultured under similar conditions.

The medium in the wells of the assay plate is then removed therefrom andthe clones attached to the well walls are washed twice with PBS(−). A5-fold diluted lysis buffer PGC50 (Toyo Ink) is added, respectively, tothe clones in the wells of the assay plate at 20 μl/well. The assayplate is left standing at room temperature for 30 minutes and is set ona luminometer LB96P (Berthold), which is equipped with an automaticsubstrate injector. Subsequently, 50 μl of the substrate solution PGL100(Toyo Ink) is automatically dispensed to the lysed clones in the assayplate to measure the luciferase activity therein with the luminometerLB96P. A plurality of clones, which exhibited a high luciferase activityare selected therefrom.

Samples of the selected clones are then cultured at 37° C. for 1 to 2weeks in the presence of 5% CO₂ using dishes having a diameter of about10 cm (Falcon) in charcoal dextran FBS/E-MEM medium.

The plasmid pRc/RSV-hPPAR γ Kozak is then introduced to the samples ofthe selected clones by a lipofection method using lipofectamine (LifeTechnologies) to provide a second round of clones. According with themanual provided with the lipofectamine, the conditions under thelipofection method included 5 hours of treatment, 7 μg/dish ofpRc/RSV-hPPAR γ Kozak and 21 μl/dish of lipofectamine. A DMSO solutioncontaining 15d prostaglandin J2, which is the natural cognate ligand ofa human normal PPAR γ, is then added to the resulting second clones sothat the concentration of 15d prostaglandin J2 in the medium is 10 nM.After culturing the second clones for 2 days, the luciferase activity ismeasured, similarly to the above, for each of the second clones. Theclone in the master plate, which provided the second clone exhibitingthe highest induction of luciferase activity, is selected as the stablytransformed DR1 cassette cell.

In this regard, the stably transformed DR1 cassette cell can be used inreporter assays with PPAR, RAR, retinoin X receptor, HNF-4, TR-2, TR-4and the like.

7. SEQUENCE FREE TEXT

SEQ ID:1 human normal ERα

SEQ ID:2 human mutant ERαK303R

SEQ ID:3 human mutant ERαS309F

SEQ ID:4 human mutant ERαG390D

SEQ ID:5 human mutant ERαM396V

SEQ ID:6 human mutant ERαG415V

SEQ ID:7 human mutant ERαG494V

SEQ ID:8 human mutant ERαK531E

SEQ ID:9 human mutant ERαS578P

SEQ ID:10 human mutant ERαG390D/S578P

SEQ ID:11 Designed oligonucleotide primer for PCR

SEQ ID:12 Designed oligonucleotide primer for PCR

SEQ ID:13 Designed oligonucleotide for mutagenesis

SEQ ID:14 Designed oligonucleotide for mutagenesis

SEQ ID:15 Designed oligonucleotide for mutagenesis

SEQ ID:16 Designed oligonucleotide for mutagenesis

SEQ ID:17 Designed oligonucleotide for mutagenesis

SEQ ID:18 Designed oligonucleotide for mutagenesis

SEQ ID:19 Designed oligonucleotide for mutagenesis

SEQ ID:20 Designed oligonucleotide for mutagenesis

SEQ ID:21 Designed oligonucleotide for mutagenesis

SEQ ID:22 Designed oligonucleotide for mutagenesis

SEQ ID:23 Designed oligonucleotide for mutagenesis

SEQ ID:24 Designed oligonucleotide for mutagenesis

SEQ ID:25 Designed oligonucleotide for mutagenesis

SEQ ID:26 Designed oligonucleotide for mutagenesis

SEQ ID:27 Designed oligonucleotide for mutagenesis

SEQ ID:28 Designed oligonucleotide for mutagenesis

SEQ ID:29 Designed oligonucleotide primer for PCR

SEQ ID:30 Designed oligonucleotide primer for PCR

SEQ ID:31 Designed oligonucleotide primer for PCR

SEQ ID:32 Designed oligonucleotide primer for PCR

SEQ ID:33 Designed oligonucleotide primer for PCR

SEQ ID:34 Designed oligonucleotide primer for PCR

SEQ ID:35 Designed oligonucleotide primer for PCR

SEQ ID:36 Designed oligonucleotide primer for PCR

SEQ ID:37 Designed oligonucleotide primer for PCR

SEQ ID:38 Designed oligonucleotide primer for PCR

SEQ ID:39 Designed oligonucleotide primer for PCR

SEQ ID:40 Designed oligonucleotide primer for PCR

SEQ ID:41 Designed oligonucleotide primer for PCR

SEQ ID:42 Designed oligonucleotide primer for PCR

SEQ ID:43 Designed oligonucleotide primer for PCR

SEQ ID:44 Designed oligonucleotide primer for PCR

SEQ ID:45 Designed oligonucleotide primer for PCR

SEQ ID:46 Designed oligonucleotide primer for PCR

SEQ ID:47 Designed oligonucleotide primer for PCR

SEQ ID:48 Designed oligonucleotide primer for PCR

SEQ ID:49 Designed oligonucleotide primer for PCR

SEQ ID:50 Designed oligonucleotide primer for PCR

SEQ ID:51 Designed oligonucleotide primer for PCR

SEQ ID:52 Designed oligonucleotide primer for PCR

SEQ ID:53 Designed oligonucleotide primer for PCR

SEQ ID:54 Designed oligonucleotide primer for PCR

SEQ ID:55 Designed oligonucleotide primer for PCR

SEQ ID:56 Designed oligonucleotide primer for PCR

SEQ ID:57 Designed oligonucleotide primer for PCR

SEQ ID:58 Designed oligonucleotide primer for PCR

SEQ ID:59 Designed oligonucleotide primer for PCR

SEQ ID:60 Designed oligonucleotide primer for PCR

SEQ ID:61 Designed oligonucleotide primer for PCR

SEQ ID:62 Designed oligonucleotide primer for PCR

SEQ ID:63 Designed oligonucleotide primer for PCR

SEQ ID:64 Designed oligonucleotide primer for PCR

SEQ ID:65 Designed oligonucleotide primer for PCR

SEQ ID:66 Designed oligonucleotide primer for PCR

SEQ ID:67 Designed oligonucleotide primer for PCR

SEQ ID:68 Designed oligonucleotide primer for PCR

SEQ ID:69 Designed oligonucleotide primer for PCR

SEQ ID:70 Designed oligonucleotide primer for PCR

SEQ ID:71 Designed oligonucleotide primer for PCR

SEQ ID:72 Designed oligonucleotide primer for PCR

SEQ ID:73 Designed oligonucleotide primer for PCR

SEQ ID:74 Designed oligonucleotide primer for PCR

SEQ ID:75 Designed oligonucleotide primer for PCR

SEQ ID:76 Designed oligonucleotide primer for PCR

SEQ ID:77 Designed oligonucleotide primer for PCR

SEQ ID:78 Designed oligonucleotide primer for PCR

SEQ ID:79 Designed oligonucleotide primer for PCR

SEQ ID:80 Designed oligonucleotide primer for PCR

SEQ ID:81 Designed oligonucleotide primer for PCR

SEQ ID:82 Designed oligonucleotide primer for PCR

SEQ ID:83 Designed oligonucleotide primer for PCR

SEQ ID:84 Designed oligonucleotide primer for PCR

SEQ ID:85 Designed oligonucleotide primer for PCR

SEQ ID:86 Designed oligonucleotide primer for PCR

SEQ ID:87 Designed oligonucleotide primer for PCR

SEQ ID:88 Designed oligonucleotide primer for PCR

SEQ ID:89 Designed oligonucleotide primer for PCR

SEQ ID:90 Designed oligonucleotide primer for PCR

SEQ ID:91 Designed oligonucleotide primer for PCR

SEQ ID:92 Designed oligonucleotide primer for PCR

SEQ ID:93 Designed oligonucleotide primer for PCR

SEQ ID:94 Designed oligonucleotide primer for PCR

SEQ ID:95 Designed oligonucleotide primer for PCR

SEQ ID:96 Designed oligonucleotide primer for PCR

SEQ ID:97 Designed oligonucleotide primer for PCR

SEQ ID:98 Designed oligonucleotide primer for PCR

SEQ ID:99 Designed oligonucleotide primer for PCR

SEQ ID:100 Designed oligonucleotide primer for PCR

SEQ ID:101 Designed oligonucleotide primer for PCR

SEQ ID:102 Designed oligonucleotide primer for PCR

SEQ ID:103 Designed oligonucleotide primer for PCR

SEQ ID:104 Designed oligonucleotide primer for PCR

SEQ ID:105 Designed oligonucleotide primer for PCR

SEQ ID:106 Designed oligonucleotide primer for PCR

SEQ ID:107 Designed oligonucleotide primer for PCR

SEQ ID:108 Designed oligonucleotide primer for PCR

SEQ ID:109 Designed oligonucleotide primer for PCR

SEQ ID:110 Designed oligonucleotide primer for PCR

SEQ ID:111 Designed oligonucleotide probe for Southern hybridization

SEQ ID:112 Designed oligonucleotide probe for Southern hybridization

SEQ ID:113 Designed oligonucleotide probe for Southern hybridization

SEQ ID:114 Designed oligonucleotide probe for Southern hybridization

SEQ ID:115 Designed oligonucleotide probe for Southern hybridization

SEQ ID:116 Designed oligonucleotide probe for Southern hybridization

SEQ ID:117 Designed oligonucleotide probe for Southern hybridization

SEQ ID:118 Designed oligonucleotide probe for Southern hybridization

SEQ ID:119 Designed oligonucleotide probe for Southern hybridization

SEQ ID:120 Designed oligonucleotide probe for Southern hybridization

SEQ ID:121 Designed oligonucleotide probe for Southern hybridization

SEQ ID:122 Designed oligonucleotide probe for Southern hybridization

SEQ ID:123 Designed oligonucleotide probe for Southern hybridization

SEQ ID:124 Designed oligonucleotide probe for Southern hybridization

SEQ ID:125 Designed oligonucleotide probe for Southern hybridization

SEQ ID:126 Designed oligonucleotide probe for Southern hybridization

SEQ ID:127 Designed oligonucleotide probe for Southern hybridization

SEQ ID:128 Designed oligonucleotide probe for Southern hybridization

SEQ ID:129 Designed oligonucleotide probe for Southern hybridization

SEQ ID:130 Designed oligonucleotide probe for Southern hybridization

SEQ ID:131 Designed oligonucleotide probe for Southern hybridization.

SEQ ID:132 Designed oligonucleotide probe for Southern hybridization

SEQ ID:133 Designed oligonucleotide probe for Southern hybridization

SEQ ID:134 Designed oligonucleotide probe for Southern hybridization

SEQ ID:135 Designed oligonucleotide probe for Southern hybridization

SEQ ID:136 Designed oligonucleotide probe for Southern hybridization

SEQ ID:137 Designed oligonucleotide probe for Southern hybridization

SEQ ID:138 Designed oligonucleotide probe for Southern hybridization

SEQ ID:139 Designed oligonucleotide probe for Southern hybridization

SEQ ID:140 Designed oligonucleotide probe for Southern hybridization

SEQ ID:141 Designed oligonucleotide probe for Southern hybridization

SEQ ID:142 Designed oligonucleotide probe for Southern hybridization

SEQ ID:143 Designed oligonucleotide probe for Southern hybridization

SEQ ID:144 Designed oligonucleotide probe for Southern hybridization

SEQ ID:145 Designed oligonucleotide probe for Southern hybridization

SEQ ID:146 Designed oligonucleotide probe for Southern hybridization

SEQ ID:147 Designed oligonucleotide probe for Southern hybridization

SEQ ID:148 Designed oligonucleotide probe for Southern hybridization

SEQ ID:149 Designed oligonucleotide probe for Southern hybridization

SEQ ID:150 Designed oligonucleotide probe for Southern hybridization

SEQ ID:151 Designed oligonucleotide primer for PCR

SEQ ID:152 Designed oligonucleotide for mutagenesis

SEQ ID:153 Designed oligonucleotide for mutagenesis

SEQ ID:154 Designed oligonucleotide for mutagenesis

SEQ ID:155 Designed oligonucleotide for mutagenesis

SEQ ID:156 Designed oligonucleotide for mutagenesis

SEQ ID:157 Designed oligonucleotide for mutagenesis

SEQ ID:158 Designed oligonucleotide primer for PCR

SEQ ID:159 Designed oligonucleotide primer for PCR

SEQ ID:160 Designed oligonucleotide primer for PCR

SEQ ID:161 Designed oligonucleotide for synthesis

SEQ ID:162 Designed oligonucleotide for synthesis

SEQ ID:163 Designed oligonucleotide for synthesis

SEQ ID:164 Designed oligonucleotide primer for PCR

SEQ ID:165 Designed oligonucleotide primer for PCR

SEQ ID:166 Designed oligonucleotide primer for PCR

SEQ ID:167 Designed oligonucleotide primer for PCR

SEQ ID:168 Designed oligonucleotide primer for PCR

SEQ ID:169 Designed oligonucleotide primer for PCR

SEQ ID:170 Designed oligonucleotide primer for PCR

SEQ ID:171 Designed oligonucleotide primer for PCR

SEQ ID:172 Designed oligonucleotide primer for PCR

SEQ ID:173 Designed oligonucleotide primer for PCR

SEQ ID:174 Designed oligonucleotide primer for PCR

SEQ ID:175 Designed oligonucleotide primer for PCR

SEQ ID:176 Designed oligonucleotide primer for PCR

SEQ ID:177 Designed oligonucleotide primer for PCR

SEQ ID:178 Designed oligonucleotide primer for PCR

SEQ ID:179 Designed oligonucleotide primer for PCR

SEQ ID:180 Designed oligonucleotide primer for PCR

SEQ ID:181 Designed oligonucleotide primer for PCR

SEQ ID:182 Designed oligonucleotide primer for PCR

SEQ ID:183 Designed oligonucleotide primer for PCR

SEQ ID:184 Designed oligonucleotide primer for PCR

SEQ ID:185 Designed oligonucleotide primer for PCR

SEQ ID:186 Designed oligonucleotide primer for PCR

SEQ ID:187 Designed oligonucleotide primer for PCR

SEQ ID:188 Designed oligonucleotide primer for PCR

SEQ ID:189 Designed oligonucleotide primer for PCR

SEQ ID:190 Designed oligonucleotide primer for PCR

SEQ ID:191 Designed oligonucleotide primer for PCR

SEQ ID:192 Designed oligonucleotide primer for PCR

SEQ ID:193 Designed oligonucleotide primer for PCR

SEQ ID:194 Designed oligonucleotide primer for PCR

SEQ ID:195 Designed oligonucleotide primer for PCR

SEQ ID:196 Designed oligonucleotide primer for PCR

SEQ ID:197 Designed oligonucleotide primer for PCR

SEQ ID:198 Designed oligonucleotide primer for PCR

SEQ ID:199 Designed oligonucleotide primer for PCR

SEQ ID:200 Designed oligonucleotide primer for PCR

SEQ ID:201 Designed oligonucleotide primer for PCR

SEQ ID:202 Designed oligonucleotide primer for PCR

SEQ ID:203 Designed oligonucleotide primer for PCR

SEQ ID:204 Designed oligonucleotide for synthesis

SEQ ID:205 Designed oligonucleotide primer for PCR

SEQ ID:206 Designed oligonucleotide primer for PCR

SEQ ID:207 Designed oligonucleotide primer for PCR

SEQ ID:208 Designed oligonucleotide primer for PCR

SEQ ID:209 Designed oligonucleotide for synthesis

SEQ ID:210 Designed oligonucleotide primer for PCR

SEQ ID:211 Designed oligonucleotide primer for PCR

SEQ ID:212 Designed oligonucleotide primer for PCR

SEQ ID:213 designed oligonucleotide for synthesis

1. An artificial cell comprising: (i) a chromosome which comprises areporter polynucleotide, wherein the reporter polynucleotide comprisesan ERE, a TATA sequence and a reporter sequence heterologous to the ERE;and (ii) a mutant ERα which has an activity for transactivation of thereporter polynucleotide, wherein in the presence of a partialanti-estrogen and E2 the activity is higher than that of ERα encoded bySEQ ID NO:1 in the presence of the partial anti-estrogen and E2, or inthe presence of a partial anti-estrogen the activity is higher than thatof ERα encoded by SEQ ID NO:1 in the presence of the partialanti-estrogen, and wherein the mutant ERα has an amino acid sequence ofan ERα comprising one or more substituted amino acids at one or morerelative positions selected from 303, 309, 390, 396, 494 and 578, or twoor more substituted amino acids at two or more relative positionsselected from 303, 309, 390, 396, 415, 494, 531 and 578, wherein therelative positions are based on a homology alignment to an amino acidsequence shown in SEQ ID NO:1.
 2. The artificial cell according to claim1, wherein the polynucleotide encoding the mutant ERα is operably linkedto a promoter and comprised by a vector.
 3. The artificial cellaccording to claim 1, wherein the partial anti-estrogen is tamoxifen,raloxifene or 4-hydroxytamoxifen.
 4. The artificial cell according toclaim 1, wherein the activity is also an activity for transactivation ofthe reporter gene which is inhibited in the presence of a pureanti-estrogen.
 5. An artificial cell comprising: (i) a chromosome whichcomprises a reporter polynucleotide, wherein the reporter polynucleotidecomprises an ERE, a TATA sequence and a reporter sequence heterologousto the ERE; and (ii) a mutant ERα which activates transcription of apolynucleotide downstream from an ERE while exposed to an anti-estrogenwhich is not antagonistic to an AF1 region of ERα encoded by SEQ ID NO:1and is antagonistic to an AF2 region of ERα encoded by SEQ ID NO:1, saidmutant ERα comprising: an amino acid sequence of an ERα comprising oneor more substituted amino acids at one or more relative positionsselected from 303, 309, 390, 396, 494 and 578, or two or moresubstituted amino acids at two or more relative positions selected from303, 309, 390, 396, 415, 494, 531 and 578, wherein the relativepositions are based on a homology alignment to an amino acid sequenceshown in SEQ ID NO:1.
 6. The artificial cell according to claim 5,wherein the mutant ERα is one which also activates transcription of thepolynucleotide downstream from an EKE while bound to E2, wherein theactivation is not inhibited by the anti-estrogen which is notantagonistic to an AF1 region of ERα encoded by SEQ ID NO:1 and isantagonistic to an AF2 region of ERα encoded by SEQ ID NO:1.
 7. Anisolated mutant ERα having: an activity for transactivation of areporter polynucleotide, the reporter polynucleotide comprising an EKE,a TATA sequence and a reporter sequence heterologous to the ERE, whereinin the presence of a partial anti-estrogen and E2 the activity is higherthan that of ERα encoded by SEQ ID NO:1 in the presence of the partialanti-estrogen and E2, or in the presence of a partial anti-estrogen theactivity is higher than that of ERα encoded by SEQ ID NO:1 in thepresence of the partial anti-estrogen; and an amino acid sequence of anERα comprising one or more substituted amino acids at one or morerelative positions selected from 303, 309, 390, 396, 494 and 578, or twoor more substituted amino acids at two or more relative positionsselected from 303, 309, 390, 396, 415, 494, 531 and 578, wherein therelative positions are based on a homology alignment to an amino acidsequence shown in SEQ ID:1.
 8. The isolated mutant ERα according toclaim 7, wherein the substituted amino acid is an arginine at relativeposition 303, a phenylalanine at relative position 309, an asparaginicacid at relative position 390, a valine at relative position 396, avaline at relative position 494 or a proline at a relative position 578,wherein the relative positions are based on a homology alignment to anamino acid sequence shown in SEQ ID:1.
 9. The isolated mutant ERαaccording to claim 7, wherein the substituted amino acid is an aminoacid other than lysine at relative position 303, an amino acid otherthan serine at relative position 309, an amino acid other than glycineat relative position 390, an amino acid other than methionine atrelative position 396, an amino acid other than glycine at relativeposition 494 and an amino acid other than serine at relative position578, wherein the relative positions are based on a homology alignment toan amino acid sequence shown in SEQ ID:1.
 10. An isolated mutant ERαhaving an amino acid sequence shown in SEQ ID:2.
 11. An isolated mutantERα having an amino acid sequence shown in SEQ ID:3.
 12. An isolatedmutant ERα having an amino acid sequence shown in SEQ ID:4.
 13. Anisolated mutant ERα having an amino acid sequence shown in SEQ ID:5. 14.An isolated mutant ERα having an amino acid sequence shown in SEQ ID:7.15. An isolated mutant ERα having an amino acid sequence shown in SEQID:9.
 16. An isolated mutant ERα having an amino acid sequence shown inSEQ ID:10.
 17. An isolated polynucleotide encoding the mutant ERα ofclaim
 7. 18. An isolated polynucleotide encoding the mutant ERα of claim10.
 19. An isolated polynucleotide encoding the mutant ERα of claim 11.20. An isolated polynucleotide encoding the mutant ERα of claim
 12. 21.An isolated polynucleotide encoding the mutant ERα of claim
 13. 22. Anisolated polynucleotide encoding the mutant ERα of claim
 14. 23. Anisolated polynucleotide encoding the mutant ERα of claim
 15. 24. Anisolated polynucleotide encoding the mutant ERα of claim
 16. 25. Avector comprising the polynucleotide of claim
 17. 26. A virus comprisingthe vector of claim 25.